M A N U A L O F B A S I C TECHNIQUES

FOR A HEALTH L A B O R AT O R Y
2nd edition

World Health Organization Geneva

The World Health Organization was established in 1948 as a specialized agency of the United Nations serving as the directing and coordinating authority for international health matters and public health. One of WHO’s constitutional functions is to provide objective and reliable information and advice in the field of human health, a responsibility that it fulfils in part through its extensive programme of publications. The Organization seeks through its publications to support national health strategies and address the most pressing public health concerns of populations around the world. To respond to the needs of Member States at all levels of development, WHO publishes practical manuals, handbooks and training material for specific categories of health workers; internationally applicable guidelines and standards; reviews and analyses of health policies, programmes and research; and state-of-the-art consensus reports that offer technical advice and recommendations for decision-makers. These books are closely tied to the Organization’s priority activities, encompassing disease prevention and control, the development of equitable health systems based on primary health care, and health promotion for individuals and communities. Progress towards better health for all also demands the global dissemination and exchange of information that draws on the knowledge and experience of all WHO’s Member countries and the collaboration of world leaders in public health and the biomedical sciences. To ensure the widest possible availability of authoritative information and guidance on health matters, WHO secures the broad international distribution of its publications and encourages their translation and adaptation. By helping to promote and protect health and prevent and control disease throughout the world, WHO’s books contribute to achieving the Organization’s principal objective – the attainment by all people of the highest possible level of health.

Contents

i

Manual of basic techniques for a health laboratory
Second edition

World Health Organization Geneva 2003

ii

Manual of basic techniques for a health laboratory

WHO Library Cataloguing-in-Publication Data Manual of basic techniques for a health laboratory. — 2nd ed. 1.Clinical laboratory techniques — handbooks ISBN 92 4 154530 5 2.Technology, Medical — handbooks 3.Manuals

(NLM classification: QY 25)

© World Health Organization 2003 All rights reserved. Publications of the World Health Organization can be obtained from Marketing and Dissemination, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel.: +41 22 791 2476; fax: +41 22 791 4857; e-mail: bookorders@who.int). Requests for permission to reproduce or translate WHO publications – whether for sale or for noncommercial distribution – should be addressed to Publications, at the above address (fax: +41 22 791 4806; e-mail: permissions@who.int). The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines for which there may not yet be full agreement. The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed or recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters. The World Health Organization does not warrant that the information contained in this publication is complete and correct and shall not be liable for any damages incurred as a result of its use.

Design by minimum graphics Typeset in Hong Kong Printed in Malta 99/12670 — SNPBest-set/Interprint — 15000

Contents

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Contents

Preface 1. Introduction 1.1 1.2 Aim of the manual Reagents and equipment 1.2.1 1.2.2 1.3 1.4 Reagents Equipment

x 1 1 1 1 1 2 2 2 2

The responsibility of laboratory workers Units of measurement 1.4.1 1.4.2 Quantities and units in the clinical laboratory SI units and names for quantities

PART I
2. Setting up a peripheral health laboratory 2.1 Plan of a peripheral medical laboratory 2.1.1 2.1.2 2.2 A one-room laboratory A two-room laboratory

9
11 11 11 12 12 13 15 17 20 20 20 22 23 24 24 27 29 32 32 33 33 33 42 45 46 46

Electricity 2.2.1 2.2.2 2.2.3 Sources of electricity Setting up simple electrical equipment What to do in case of failure of electrical equipment

2.3

Plumbing: simple procedures 2.3.1 2.3.2 2.3.3 Tools and materials Taps Sink traps

2.4

Water for laboratory use 2.4.1 2.4.2 2.4.3 2.4.4 Clean water Distilled water Demineralized water Buffered water

2.5

Equipment 2.5.1 2.5.2 2.5.3 2.5.4 2.5.5 2.5.6 Essential laboratory instruments Additional items Equipment and supplies Making glass equipment Specimen containers Storage, stocktaking and ordering supplies

2.6

Registration of specimens and preparation of monthly reports 2.6.1 Registration of specimens

iii

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Manual of basic techniques for a health laboratory

2.6.2

Preparation of monthly reports

47 53 53 53 58 61 63 64 64 66 67 67 68 69 69 69 70 71 73 73 75 77 77 77 77 81 83 83 85 90 90 90 91 91 91 of biopsy specimens for 95 96 97 98 101 102

3. General laboratory procedures 3.1 Use of a microscope 3.1.1 3.1.2 3.1.3 3.1.4 3.1.5 3.1.6 3.2 Components of a microscope Setting up the microscope Focusing the objective Use of an ocular micrometer Dark-field microscopy Routine maintenance

Weighing: use of laboratory balances 3.2.1 3.2.2 3.2.3 3.2.4 Sensitivity of a balance Open two-pan balance Analytical balance Dispensary balance

3.3

Centrifugation 3.3.1 3.3.2 3.3.3 Principle Types of centrifuge Instructions for use

3.4

Measurement and dispensing of liquids 3.4.1 3.4.2 3.4.3 3.4.4 Pipettes Volumetric flasks Burettes Graduated conical glasses

3.5

Cleaning, disinfection and sterilization 3.5.1 3.5.2 3.5.3 3.5.4 3.5.5 Cleaning glassware and reusable syringes and needles Cleaning non-disposable specimen containers Cleaning and maintenance of other laboratory equipment Disinfectants Sterilization

3.6

Disposal of laboratory waste 3.6.1 3.6.2 3.6.3 Disposal of specimens and contaminated material Incineration of disposable materials Burial of disposable materials

3.7

Dispatch of specimens to a reference laboratory 3.7.1 3.7.2 Packing specimens for dispatch Fixation and dispatch histopathological examination

3.8

Safety in the laboratory 3.8.1 3.8.2 Precautions to prevent accidents First aid in laboratory accidents

3.9

Quality assurance in the laboratory 3.9.1 Specimen collection

PART II
4. Parasitology 4.1 4.2 Introduction Examination of stool specimens for parasites

103
105 105 107

Contents

v

4.2.1 4.2.2 4.2.3 4.2.4 4.3

Collection of specimens Visual examination Microscopic examination Dispatch of stools for detection of parasites

107 107 107 109 111 111 118 125 126 146 152 152

Intestinal protozoa 4.3.1 4.3.2 Identification of motile forms (trophozoites) Identification of cysts

4.4

Intestinal helminths 4.4.1 4.4.2 Identification of eggs Identification of adult helminths

4.5

Techniques for concentrating parasites 4.5.1 4.5.2 4.5.3 4.5.4 Flotation technique using sodium chloride solution (Willis) Formaldehyde–ether (Allen & Ridley) sedimentation

technique 153 154 Strongyloides 156 157 157 157 158 159 159 159 172 182 194 197 197 197 197 197 198 199 199 199

Formaldehyde–detergent sedimentation technique Sedimentation technique stercoralis (Harada–Mori) for larvae of

4.6

Chemical test for occult blood in stools 4.6.1 4.6.2 4.6.3 4.6.4 Principle Materials and reagents Method Results

4.7

Parasites of the blood and skin 4.7.1 4.7.2 4.7.3 4.7.4 Filariae Plasmodium spp. Trypanosoma spp. Leishmania spp.

5. Bacteriology 5.1 5.2 Introduction Preparation and fixation of smears 5.2.1 5.2.2 5.2.3 5.2.4 5.3 Principle Materials and reagents Preparation of smears Fixation of smears

Staining techniques 5.3.1 5.3.2 5.3.3 5.3.4 5.3.5 Gram staining Staining with Albert stain Corynebacterium diphtheriae) (for the detection

of 201

Staining with Ziehl–Neelsen stain (for the detection of acid-fast bacilli) 202 Staining with Wayson stain (for the detection of Yersinia pestis) 203 Staining with Loeffler methylene blue (for the detection of Bacillus anthracis) 204 204 205 205 206 206

5.4

Examination of sputum specimens and throat swabs 5.4.1 5.4.2 5.4.3 5.4.4 Materials and reagents Method Microscopic examination Dispatch of specimens for culture

2 5.11. Mycology 6.7.2 5.2.1 5.3.1 5.4 Materials and reagents Method Macroscopic examination Microscopic examination 5.1 5.6.3 5.11 Examination of pus for Bacillus anthracis 5.1 6.3 Materials and reagents Method Microscopic examination 5.6.9.10.12 Examination of skin Mycobacterium leprae 5.3 Examination of skin for pityriasis versicolor 6.1 5.8.7 Examination of semen specimens 5.3 5.2 5.2 Materials and reagents Method Examination of pus for mycetoma 6.3.vi Manual of basic techniques for a health laboratory 5.5.3 for 220 220 221 223 225 225 225 225 226 227 227 227 227 228 Materials and reagents Method Microscopic examination 6.3 Materials and reagents Method Microscopic examination 5.5 Examination of urogenital specimens for gonorrhoea 5.1 6.2 5.3 Materials and reagents Method Microscopic examination smears and nasal scrapings 5.1.5.1 Examination of skin and hair for fungi 6.12.3 5.6.9.7.4 Materials and reagents Method Microscopic examination Dispatch of specimens for culture 5.11.9.1 6.1 5.11.12.1 5.2 5.8.2 5.7.2 5.10 Examination of aspirates.1 5.7.10.8 Examination of vaginal discharge 5.1.1 5.8.5.5.2.2 5.10.6 Examination of genital specimens for syphilis 5.12.3 Materials and reagents Method Microscopic examination 5. exudates and effusions 5.2 Materials and reagents Method 6.2 Materials and reagents Method .4 Materials and reagents Method Microscopic examination Dispatch of specimens for culture 207 207 207 208 209 209 210 210 211 211 211 212 212 212 215 215 215 215 216 216 216 216 216 218 218 218 219 219 219 220 220 5.9 Examination of watery stool specimens 5.9.2 6.

5 Estimation of the erythrocyte number concentration .4 Estimation of the erythrocyte volume fraction 9.1 9.1 Types of blood cell 9.1 9.2.1 8.2.2 Erythrocytes Leukocytes Thrombocytes Collection of blood specimens 9.1 9.2 Haemiglobincyanide photometric method Alkaline haematin D method 9.8 7.2 Micro-scale method Macro-scale method 9. Haematology 9.3.7 7.4 8.2 9.3.2.1.2 8.2.1 9.6 7.Contents vii PART III 7.2.2 7.2.4.3 8.2.2 8.1 7.2.1 Collection of urine specimens 7.1.1.5 8.3 7.3 Estimation of the haemoglobin concentration 9.5 7.6 8.3.4 Precautions Direct examination Microscopic examination Determination of glucose concentration Determination of protein concentration Summary Dispatch of CSF specimens for culture 8.3 Common reasons for investigation of CSF Collection of CSF specimens Examination of CSF specimens 8.9 Appearance Testing for the presence of blood Measuring the pH Detection of glucose Detection and estimation of protein Detection of ketone bodies Detection of abnormal elements Detection of Schistosoma haematobium infection Detection of bacteria 8.1.3 9.3.1 8.3.3.3.4.3 Principle Materials and reagents Method 9.2.1.2.1 7.4.2 Types of urine specimen Preservation of urine specimens 231 233 233 233 234 234 234 234 235 236 236 239 240 249 251 255 255 255 255 255 256 257 261 262 263 263 263 Examination of urine specimens 7.2 7.2.4 7.1 8.2. Examination of cerebrospinal fluid (CSF) 8.2 9.3.4. Examination of urine 7.2 Materials and reagents Method using Stuart transport medium (for the isolation of Neisseria meningitidis) 264 265 265 265 265 266 267 267 267 267 271 271 276 279 280 286 287 9.

1.2 9.12.3 9.4 Principle Materials Method Results 9.4 Principle Materials and reagents Method Microscopic examination 9.4 Principle Materials and reagents Method Results 9.14 Determination of the thrombocyte number concentration 9.12.10.1 9.7.1 9.10.8.2 9.1 9.7 Measurement of the erythrocyte sedimentation rate 9.10.1 9.12.13.2 9.4 Principle Materials and reagents Method Results 9.1 Estimation method 10.7.6. Blood chemistry 10.11.4 Principle Materials and reagents Method Microscopic examination 9.3 9.11 Test for sickle-cell anaemia 9.13 Determination of the leukocyte type number fraction 9.14.11.1 9.7.4 Principle Materials and reagents Method Results 288 288 288 289 291 292 292 292 292 293 295 295 295 295 296 297 297 297 297 298 299 299 299 300 305 314 314 314 315 315 316 316 316 317 318 319 319 319 320 321 321 321 322 9.9 Observation of clot retraction and measurement of lysis time 9.1.9.10 Preparation and staining of thin blood films 9.13.2 9.viii Manual of basic techniques for a health laboratory 9.3 9.2 9.9.3 Principle Materials Microscopic examination 9.3 9.13.7.1 9.8.12 Determination of the reticulocyte number concentration/fraction 9.12.2 of glucose concentration in blood: o-toluidine 322 322 322 Principle Materials and reagents .2 9.10.8 Measurement of the bleeding time: Duke method 9.11.1 9.8.2 Materials Microscopic examination 10.2 9.3 9.2 9.6.1 9.11.8.1 10.1 9.6.9.4 Principle Materials and reagents Method Microscopic examination 9.9.3 9.6.3 9.14.6 Estimation of the leukocyte number concentration 9.

1 11.9 Dipstick test for falciparum malaria 11.5.9.1.2.2 Antibodies Antigens Antigen–antibody interactions Principle of immunochemical techniques 11.Contents ix 10.7.2 ELISA for hepatitis B surface antigen Dipstick test for hepatitis B surface antigen 11.2.2 ELISA Dipstick test 11.3 10.2.4.10.9.3 10.8 Tests for hepatitis virus infection 11.1 11.2 TPHA test Annex: Reagents and their preparation Index .1 Introduction to immunology 11.4 Tests for the determination of anti-streptolysin O antibodies 11.1.8.3 11.2 Materials and reagents Method 11.10 Tests for syphilis infection 11.2 Materials and reagents Method 11.8.1.1 11.6.1 11.2 Method Results 322 324 Estimation of urea concentration in blood: diacetyl monoxime/ thiosemicarbazide method 325 10.2 10.2 Primary binding tests Secondary binding tests 11.1 11.7 Tests for the determination of HIV antibodies 11.3.6 Quantitative determination immunodiffusion 11.5 Determination of b-human chorionic gonadotropin (b-hCG) in urine by the agglutination inhibition technique 339 11.5. Immunological and serological techniques 11.4 10.1.2 Materials and reagents Method of IgA.1 11.2 Anti-streptolysin O test (ASOT) Latex agglutination 11.1 10.3.4.2.10.1 11.1 11.2 11.4 Principle Materials and reagents Method Results 325 325 326 327 328 328 328 329 330 330 330 332 11.1 11.6.2.2.7. IgG and IgM by 339 339 radial 339 339 340 341 341 342 342 343 344 344 344 345 346 347 348 350 369 11.2 Materials and reagents Method 336 336 336 336 338 11.3 Determination of rheumatoid factors by the latex-agglutination technique 336 11.1 RPR test 11.1.

particular attention has been paid to the low cost.H. WHO expresses its thanks to all those who have assisted in the revision of this manual. It is intended mainly for the use of laboratory personnel in developing countries during their training and thereafter in their work. major revisions having been carried out by Dr K. x . In the selection of techniques. Dr C.x Manual of basic techniques for a health laboratory Preface This book is a revised edition of the Manual of basic techniques for a health laboratory (WHO.C. and some obsolete procedures have been replaced by more up-to-date techniques. The revision was necessary because of new procedures and technology that have been developed since the previous edition and that have proved to be useful to small laboratories in developing countries. The original objective of the manual remains unchanged. Heuck and Mr A. reliability and simplicity of the methods and to the availability of resources in small laboratories. Moody. The procedures have been included in the relevant sections of the manual. Engbaek. 1980).

Such procedures include the following: — the examination of stools for helminth eggs. — the examination of blood for determination of the white cell (leukocyte) type number fraction (differential leukocyte count) — the examination of blood for determination of the glucose concentration. It is designed particularly for use in peripheral laboratories in such countries (i.2 Equipment The items required for each technique are listed at the beginning of the corresponding section.2.1 Reagents Each reagent has been given a number. When certain articles are not available. 1. The language used has been kept as simple as possible although common technical terms are employed when necessary.2 Reagents and equipment 1. 1. For example. their composition. The composition of crystal violet and the method of preparing it are given in the alphabetical list of reagents (see Annex). modified Hucker (reagent no. 18). — the examination of urine for bile pigments. The manual describes examination procedures that can be carried out with a microscope or other simple apparatus. For example. in small or medium-sized laboratories attached to regional hospitals) and in dispensaries and rural health centres where the laboratory technician often has to work alone. the technician should find an appropriate substitute. racks for test1 .5. — the examination of sputum for tubercle bacilli.2. a laboratory in a rural health centre may not be able to carry out certain blood chemistry or serological tests. A list of the apparatus needed to equip a laboratory capable of carrying out all the examinations described in this manual can be found in section 2. Introduction 1 1.e. — the examination of blood for malaria parasites. empty bottles that formerly contained antibiotics for injection (“penicillin bottles”) and other drug containers can be kept.1. The reagents required and their numbers are indicated in the description of each technique.1 Aim of the manual This manual is intended for use mainly in medical laboratories in developing countries. for example. An alphabetical list of all the reagents used. Introduction 1. methods of preparation and storage requirements appears in the Annex at the end of the manual. Some laboratories may not be able to perform all the procedures described. The intention is to provide an account of basic laboratory techniques that are useful to peripheral laboratories and can be carried out with a limited range of basic equipment. with the numbers assigned to them. one of the reagents needed for Gram staining is crystal violet.

1 Quantities and units in the clinical laboratory Almost all your work in the laboratory will involve making measurements of quantities and using units for reporting the results of those measurements. and new names were introduced to replace them. These units have been used to an increasing extent in the sciences.3 The responsibility of laboratory workers Laboratory workers carry out laboratory examinations to provide information for clinical staff in order to benefit patients. 1. They therefore play an important role in helping patients to get better. temperature and electric current are quantities. 1. At the same time. 1. Note that the word “quantity” has two meanings. whereas the standards in which they are measured are units.4. but most of them were introduced into medicine only after 1960. Since then this system has been gradually expanded. must regard this information as strictly confidential. these are also included and the relationship between the two is explained. To accompany the introduction of SI units. Laboratory workers. Units of measurement that form part of this system are called “SI units”. and empty tins can be used to make waterbaths.2 Manual of basic techniques for a health laboratory tubes and slides can be made locally. . in the course of their work. medical scientists prepared a systematic list of names for quantities. you should thoroughly understand: — the quantities you measure. Every laboratory worker handling clinical materials must maintain these standards. the traditional names were inaccurate. speed.4 Units of measurement In the laboratory you will work extensively with both quantities and units of measurement. length. However. In scientific usage height. they gain a lot of information about patients and their illnesses. since 1901 (long before they were called SI units). like clinical staff. Some of these names are the same as the traditional ones. in other cases. and it is important to understand the difference between them. — the units that are used to measure the quantities. however. 1. and in 1960 it was given the name “Système international d’Unités” (International System of Units) and the international abbreviation “SI”. misleading or ambiguous. the scientific meaning just defined and the everyday meaning “amount of ”. especially chemistry and physics. only the clinical staff who request the examinations should receive the reports on them. In most countries there are high moral and professional standards of behaviour for clinical staff and qualified laboratory personnel.2 SI units and names for quantities A simple standardized set of units of measurement has been the goal of scientists for almost two centuries. When patients enquire about test results they should be told to ask the clinical staff.4. Any measurable physical property is called a quantity. The metric system was introduced in 1901. since traditional units and names for quantities are still used in some laboratories. Since the health — and even the life — of a patient may depend on the care with which you make a measurement and the way in which you report the results. This manual uses SI units and the currently accepted names for quantities. — the names that are given to those quantities.

they do not do so in relation to their mass. In some cases. although the quantity names “mass” and “amount of substance” and the unit name “mole” may need explanation. Table 1. SI units used in this manual All SI units are based on seven SI base units. For example: sodium hydrochloric sodium carbon + Æ + + water bicarbonate acid chloride dioxide In this reaction 1 kg (1 kilogram) of sodium bicarbonate does not react with 1 kg of hydrochloric acid.) “Amount of substance” and its unit. mole. Mass. Mass is the correct term for what is commonly called “weight”. is independent of the earth’s gravitational attraction. are important terms in medicine and they will affect your work in the laboratory more than any other quantities or SI units. (There is a technical meaning of the term “weight”: it is a measure of the force with which the earth’s gravity attracts a given mass. the unit of volume is metre ¥ metre ¥ metre = metre cubed or cubic metre.1 SI base units used in this manual Quantity Length Mass Time Amount of substance Unit name metre kilogram second mole Symbol m kg s mol The first three of these units will be familiar to you. Only four of them are used in this manual. and the unit of speed is metre divided by second = metre per second. 1 mol (1 mole) of sodium bicarbonate reacts with 1 mol of hydrochloric acid. All the SI derived units are obtained in this simple way. therefore gives a measure of equivalent amounts of two or more different substances (use of mass units does not). however. Whenever chemical substances interact. furthermore. Most of the SI units are called SI derived units. Some common SI derived units are shown in Table 1. Introduction 3 The following section gives a brief description of the SI units and of the quantity names that are used in this manual. When two or more chemical substances react together. These are obtained by combining the SI base units (by multiplication or division) as appropriate.2. on the other hand. in fact.1. we speak of measuring a mass as “weighing”. Use of the mole. Table 1.2 SI derived units used in this manual Quantity Area Volume Speed Unit name square metre cubic metre metre per second Symbol m2 m3 m/s or ms-1 The unit of area is metre ¥ metre = metre squared or square metre. it is necessary to multiply and . The two terms are mixed up in everyday usage. they do so in relation to their relative molecular mass (the new name for what used to be called “molecular weight”). they are listed in Table 1.1. which is based on the relative molecular mass.

has been approved for use as a special name for the cubic decimetre. Table 1. Now each different way of expressing concentration has its own special name.01 metre (0. on graduated glassware) volumes . To avoid this difficulty such units are given special names. one-thousandth of a cubic metre). and a cubic decimetre is 0. The name “litre”. 1 centimetre (1 cm) = 0.000 001 or 10 ) Divide by 1000 million (¥ 0.01 m or 10-2 m). For example. you may encounter (for example. To overcome this difficulty. The SI prefixes used in this manual are listed in Table 1. 1 millimetre (1 mm) = 0. are used mainly for measuring relatively small volumes of liquids and sometimes gases.1 ¥ 0. and the resulting expression becomes very cumbersome. the metre is far too large to be convenient for measurement of the diameter of a red blood cell (erythrocyte). These prefixes have the same meaning when they are applied to any other unit. that is. However.001 or 10-3) Divide by 1 000 000 (¥ 0. The litre is the unit used in the clinical laboratory for reporting all concentrations and related quantities. You are probably familiar with this unit of volume. which when added to the name of a unit multiply or divide that unit by a certain factor. A submultiple of the cubic metre is therefore used. For example. although not part of the SI.001 m3 (or 10-3 m3. measurements would be difficult because these units are too large or too small for many purposes. Traditionally all of these were called simply “concentration”. but this is far too large to be convenient for measurements of body fluids. The prefix “deci” was not listed above because it is not used in this manual.000 001 metre (0. the SI incorporates a series of prefixes. but it means division by 10 (or multiplication by 0. such as the millilitre (ml). and may have been surprised that it has not already been mentioned. A decimetre is therefore 0. If the SI base units and derived units were the only ones available.000 000 001 or 10-9) -6 6 Prefix mega kilo centi milli micro nano Symbol M k c m m n For example.001 metre (0. Most of these names are used to describe concentration and related quantities. Units for measurement of concentration The difficulty with concentration is that it can be expressed in different ways. Before these names can be described.01 or 10-2) Divide by 1000 (¥ 0.3 SI prefixes Factor Multiply by 1 000 000 or 1 million (¥ 10 ) Multiply by 1000 (¥ 103) Divide by 100 (¥ 0.1 m3 = 0. called SI prefixes. giving decimal multiples or submultiples of the unit. Quantity names used in this manual Certain names for quantities were introduced to accompany the change to SI units. 1 kilometre (1 km) = 1000 metres (1000 m). volumes of solids and large volumes of liquids and gases are usually measured in terms of the cubic metre or one of its multiples or submultiples. it is necessary to explain the unit of volume called the “litre” (l). The SI derived unit of volume is the cubic metre. the unit of pressure is kilogram divided by (metre ¥ second ¥ second).1 ¥ 0. for example.001 m or 10-3 m). the unit of pressure is called the pascal. This is because the litre is not an SI unit.1 m.000 001 m or 10-6 m). The litre and its submultiples. which was misleading.1 or 10-1).3. and 1 micrometre (1 mm) = 0.4 Manual of basic techniques for a health laboratory divide several times. the cubic decimetre.

The mass of dissolved salt divided by the volume of solution is called the mass concentration. For example. micromole.g. The unit in which it is measured is gram (or milligram.000 001 0.25 one-quarter. In the traditional system mass concentration was used almost exclusively. etc. Having explained the litre. sometimes “per 100 ml” (0. we can now return to the names for different ways of expressing concentration.01 0. For particles or entities that are not dissolved. the number of moles of salt) contained in the solution divided by the volume of the solution is called the amount of substance concentration. In the SI mass concentration is rarely used. suppose that we have a solution of salt.000 001 Equivalent in millilitres (ml) 1000 100 10 1 0. When SI units are used all concentrations are expressed in terms of substance concentration wherever possible. only this time the amount of dissolved salt is expressed in terms of the “amount of substance”.) per litre. marked in terms of submultiples of the cubic metre. for short.) per litre.1 litre). Now suppose that we have another solution of salt. and we must have a way of expressing the number of cells in each litre of blood. The amount of substance of salt (that is.000 01 0. The unit in which substance concentration is measured is mole (or millimole.1. the blood contains many different kinds of cell. mass concentration was not. However. A more general definition of mass concentration is “the mass of a given component (e. making for considerable confusion. or. in the traditional system. always expressed in terms of “per litre”. the substance concentration.001 0.001 10 cm3 cm3 mm3 centilitrea millilitre microlitre Seldom used in the laboratory.1 one-tenth. 0. This use of substance concentration instead of mass concentration is the most important difference between the use of SI units and the use of traditional units. it is used only for substances such as proteins whose relative molecular mass is uncertain. Unity or 1. but it is really very simple.001 0. 0.5 represents one-half. which is defined as “the number of specified particles or entities in a mixture divided by the volume of the mixture”. five kinds of leukocyte occur in the blood. 0. The equivalent submultiples of the cubic metre and of the litre are listed in Table 1.4 SI derived units of volume Unit name Cubic decimetre — — Cubic centimetre Cubic millimetre a Symbol dm3 100 cm 3 Equivalent in cubic metres (m3) 0.0 represents the whole.0001 0. These cells are suspended in the blood. a dissolved substance) divided by the volume of solution”. For example. First.2 one-fifth. In this case the quantity name is the number concentration. Different countries (and even different laboratories in the same country) followed different practices.0 (unity). At first sight this may seem a little confusing. The . Sometimes “per litre” was used. and so on. Introduction 5 Table 1. and sometimes “per millilitre”. a different quantity must be used. the fraction of the total number that is accounted for by cells of that type. This quantity is called the number fraction. 0. and it is expressed as a fraction of 1. In the traditional system number concentration was called a “count” and it was expressed in the unit “number per cubic millimetre”.1 0. microgram.000 000 001 Unit name litre decilitre a Symbol l dl cl ml ml Equivalent in litres (l) 1 0. etc. Sometimes the quantity that is of concern is not the actual number of cells per litre (number concentration) but the proportion of cells of a given type — that is.4. The unit in which number concentration is measured is number per litre.

5 lists metric and traditional quantity names and units. (If you add these fractions.5% ¥ 10 = 5 ¥ 10-3 12 ¥ 10-3 ¥ 0. 0.01 = erythrocyte volume fraction 0.4 ¥ 100 = packed cell volume 40% Leukocyte number concentration (blood) (see section 9. ¥ 109/l reticulocyte count no.3.001 = 86.5 ¥ 109/l ¥ 1000 = 7500/mm3 No conversion factor: 27/mm3 = 27 ¥ 106/l 25 ¥ 106/l = 25/mm3 Lymphocytes 33% ¥ 0. Table 1.5 million/mm3 = 4.13 and 8. was called “packed cell volume” (which was misleading because it did not specify what kind of cell was measured and because it was reported as a percentage.g. 0.33 Lymphocyte number fraction 0.35. Another quantity that is expressed as a fraction of 1.02.08 and 0. for example.0 million/mm3 Packed cell volume 38% ¥ 0. ¥ 10-3 reticulocyte count % ‰ ./mm3 1 differential leukocyte count (e.2% 5‰ = 5 ¥ 10-3 12 ¥ 10-3 = 12‰ 1 packed cell volume (haematocrit) % no. with conversion factors.12) Reticulocyte number fractionb (see section 9.45. you will find that the total is 1.5 ¥ 1012/l 5.12) no.0 ¥ 109/l 91.0 ¥ 1012/l = 5.08 was reported as 8%.5 ¥ 109/l ¥ 1000 = 91 500/mm3 0. ¥ 1012/l Traditional quantity name erythrocyte count Traditional unit million/mm3 Conversion factors and examplesa No conversion factor: 4. each different volume fraction had a different name. The erythrocyte volume fraction is important for the diagnosis of many diseases and you will often measure it in the laboratory./mm3 8000/mm3 ¥ 0. the erythrocyte volume fraction is 450/1000 = 0.0 — the whole. Erythrocyte volume fraction.5 was reported as 50%. lymphocytes) % no. For example.6 Manual of basic techniques for a health laboratory number fraction of each type might be 0. they are both ratios. This is defined as the volume of a specified component of a mixture divided by the total volume of the mixture. see sections 9. ¥ 109/l leukocyte count (blood) leukocyte count (CSF) no. From this you will see that percentage divided by 100 gives the number fraction.3) Leukocyte type number fraction (blood and CSF) (e. Table 1.33 ¥ 100 = lymphocytes 33% no.01 = lymphocyte number fraction 0.4) SI unit no. if the total volume occupied by all the erythrocytes in 1 litre (1000 ml) of blood is 450 ml. ¥ 106/l no.3.001 = 8. and a number fraction of 0. a number fraction of 0. In the traditional system volume fraction had no special name: instead.0 is the volume fraction.10. 0. lymphocyte number fraction.5 Metric and traditional quantity names and units Quantity name Erythrocyte number concentration (see section 9. For example.38 Erythrocyte volume fraction 0.5) Erythrocyte volume fraction (see section 9.) In the traditional system this quantity had no name and results were reported as percentages instead of fractions. From the above explanation you will see that number fraction is “number per number” and volume fraction is “volume per volume” — that is.0 ¥ 109/l 7. not as a volume).3) Reticulocyte number concentration (see section 9.45.1 = 1.6) Leukocyte number concentration (CSF) (see section 8./mm3 86 000/mm3 ¥ 0.g.

7 g/100 ml ¥ 0.5 mmol/l 4.g.01 = 0.4) Protein.5) mmol/l %e g/l %e 35% ¥ 10 = 350 g/l 298 g/l ¥ 0.2 mmol/l ¥ 18.3. mass concentrationc urea nitrogen.5 g/100 ml g/l haemoglobin.9 mmol/l ¥ 6.14) Glucose. and then the conversion from SI into traditional units.5 (cont. ¥ 109/l Traditional quantity name platelet count Traditional unit no. mass concentrationc mean corpuscular haemoglobin concentration (i. mass concentration (see section 9.) Quantity name Thrombocyte number concentration (see section 9.e.25 g/l 0. each of the conversion factors listed must be multiplied or divided by 100.1 = 29.8% g/l mg/100 ml g/l 25 mg/100 ml ¥ 0.2) mmol/l urea.02 = 75. mass concentrationc g/100 ml 14. but the term “mass concentration” was not usually used.611 = 35. In this case.357 = urea 2.4 mg/100 ml urea nitrogen 7 mg/100 ml ¥ 0.7 mmol/l 22 mmol/l ¥ 1. substance concentration (blood and CSF) (see sections 10. but as a fraction of 1000.354 0. Introduction 7 Table 1. substance concentration (see section 9.3) SI unit no. The conversion factor is underlined.4) Haemoglobin (Fe).1.4) Mean erythrocyte haemoglobin mass concentration (see section 9.5 mmol/l Urea.3) Mean erythrocyte haemoglobin (Fe) substance concentration (see section 9.e mass concentration mg/100 ml mg/100 ml CSF: cerebrospinal fluid. mass concentrationc (blood and CSF) haemoglobin.01611 = 0.e.7 mmol/l 22 mmol/l ¥ 0.31 g/l ¥ 100 = 31 mg/100 ml No change 15 mg/100 ml ¥ 0.3. b In this case. c Mass concentration is what was measured.9 g/100 ml 35% ¥ 0. mass concentration)d mean corpuscular haemoglobin concentration (i.621 = Hb(Fe) 8.1 = 13.001 = 220 ¥ 109/l 250 ¥ 109/l ¥ 1000 = 250 000/mm3 81 mg/100 ml ¥ 0.35 instead of 35%.001 = 0. 0.0555 = 4.35 ¥ 62. in order to avoid inconveniently small numerical values.298 e In the traditional system urea was sometimes reported in terms of urea and sometimes in terms of urea nitrogen (i.61 = Hb 14. as in the following examples: 0. substance concentration (blood) (see section 10.7 mg/ 100 ml Hb 13. d Mean corpuscular haemoglobin concentration was sometimes expressed as a decimal fraction rather than a percentage.35 ¥ 1000 = 350 g/l 298 g/l ¥ 0.01 = 17.621 = 21. mass concentrationc mg/100 ml mmol/l g/100 ml Haemoglobin.167 = 2. mass concentration) protein. the number fraction is reported not as a fraction of 1.5 mmol/l 2.1 = 21./mm3 Conversion factors and examplesa 220 000/mm3 ¥ 0.4% mmol/l glucose. the nitrogen content of the urea).5 mmol/l Hb(Fe) 9 mmol/l ¥ 1. e. mass concentration (CSF) (see section 8. a The examples show first the conversion of actual numerical values in traditional units into values in SI units. .8 g/100 ml ¥ 10 = 148 g/l 139 g/l ¥ 0.1 and 8.e.

8 Manual of basic techniques for a health laboratory .

Setting up a peripheral health laboratory 9 Part I .2.

10 Manual of basic techniques for a health laboratory .

The plan is limited to one room.1.1 Plan of a peripheral medical laboratory 2.2 indicates another possible arrangement of a peripheral laboratory. It can obviously be modified to suit different circumstances. Setting up a peripheral health laboratory 2.1 Plan for a one-room laboratory 11 .1 A one-room laboratory Figure 2. 2. Fig.2. Setting up a peripheral health laboratory 11 2.1 sets out the possible arrangement of a peripheral medical laboratory attached to a health centre. It shows a laboratory suitable for carrying out some or all of the techniques described in the manual. since often this is all the space that is available for the laboratory. Figure 2. The room should measure at least 5 m ¥ 6 m.

11: glassware shelf. 7: electric centrifuge. 8: syphilis serology and biochemistry area. 6: waterbath. it is recommended that the second be used for washing and sterilization. 19: record-keeping area.2 Electricity A reliable energy supply should be available to ensure continuity of the work in a laboratory. 16: sinks. 20: area for examination of stool specimens. Dirty and/or contaminated material should be removed from the laboratory working area as quickly as possible.1. 12: balance. 18: bed for patients. 9: reagent refrigerator. 4: haematology area. 23: gas bottle. 14: area for examination of sputum specimens. 2. 2. 21: area for examination of urine specimens.2 A two-room laboratory If two rooms are available. 22: area for reception of specimens. both for the safety of the workers and to avoid errors and cross-contamination. 5: colorimeter. 3: microscopes. 15: Bunsen burner. The energy can be provided from the following sources: . 2: hand-operated centrifuge.12 Manual of basic techniques for a health laboratory Fig. 10: reagent shelf. 13: staining box.2 Alternative plan for a one-room laboratory 1: outpatient’s table. 17: waste sink. 2.

2. Setting up a peripheral health laboratory

13

— mains electricity supply — generators — solar energy supply system. Remote laboratories often have problems in ensuring a continuous supply of electrical power and may need to generate electricity by using a local generator or a solar energy supply system.

2.2.1 Sources of electricity
Generators Electrical energy can be provided by a fuel generator. It is possible to use the combustion engine of a motor car or a purpose-built generator. A purpose-built generator produces an alternating current of 110 volts (V) or 220 V and can usually generate more energy than a car engine. A car engine provides a direct current of 12 V or 24 V, which can be fed into rechargeable batteries (see below). The type of current available will limit the selection of laboratory equipment; for example, an instrument that requires direct current can be supplied with energy from: — batteries — a direct current network with a transformer — an alternating current network with a converter. The installation of a direct current network is simple and it is safe to operate. However, for instruments that require a low-voltage (6 V, 12 V or 24 V) direct current, the high voltage produced from the direct current network must be converted by means of a transformer. Alternatively, for instruments that require alternating current (110 V, 220 V or 240 V), the direct current must be converted into alternating current by means of an inverter. Inverters are heavy and expensive and significant energy losses occur in the conversion process. It is therefore preferable to use either direct current or alternating current appliances, depending on your supply, and avoid the need for conversion. If no generator is available or if a mains power supply is accessible, but the electrical current fluctuates or is prone to frequent breakdowns, a solar energy supply may be preferable (see below). Solar energy supply systems (photovoltaic systems) A laboratory with a few instruments with low energy requirements can work with a small energy supply. For laboratories located in remote areas, a solar energy supply system may be more suitable than a generator since there are no problems of fuel supplies and it can be easily maintained. A solar energy supply system has three components: — solar panel(s) — an electronic charge regulator — batteries. Solar panels Two different types of solar panel are commercially available: — panels with cells of crystalline silicon — panels with cells of amorphous silicon. Amorphous silicon panels are less expensive, but produce solar energy less efficiently than crystalline silicon panels.

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Manual of basic techniques for a health laboratory

Solar panels must be installed so that they are exposed to direct light, since shade reduces the efficiency of energy production. They should be inclined at an angle of 15°. The underside of the panel must be freely ventilated. The minimum distance of the underside of the panel from the surface of the supporting construction must be more than 5 cm to avoid heating of the panel, which would reduce the efficiency of energy production. Electronic charge regulators A charge regulator controls the charging and discharging of the batteries automatically. When the battery voltage falls below a threshold value during discharge, the laboratory instrument will be disconnected from the battery. On the other hand, if the voltage increases above a threshold value (e.g. when the battery is recharged), the solar panel will be disconnected from the battery. A good charge regulator adapts the maximal voltage of the battery to the change in the temperature of the ambient environment. This prevents the loss of water in the battery by evaporation. It is important to keep a spare charge regulator in stock in case of breakdown. The charge regulator chosen should be stable under tropical conditions. It is advisable to choose a charge regulator with an integrated digital display that allows the battery charge to be monitored easily. Batteries Lead batteries Solar energy systems require rechargeable batteries, which may be either lead or nickel–cadmium (Ni–Cd) batteries. Lead batteries are preferred and many types are available commercially (see Table 2.1). High-efficiency batteries have practical advantages, although they are more expensive than normal batteries. When purchasing batteries choose 12 V batteries with the highest capacity (1000 ampere-hours (Ah)). Several types of maintenance-free lead batteries are commercially available, but they are expensive and less efficient than those that require maintenance. The development of this type of battery is still in progress; it has not been thoroughly tested in tropical climates. Therefore, the maintenance-free batteries are not recommended. Transport of lead batteries Lead batteries should be emptied before being transported. It is important to remember that if lead batteries are to be transported by air they must be empty of electrolyte solution, which should be replaced on arrival at the destination. Table 2.1 Specifications for batteries used for solar power supply
Specification Nickel–cadmium Type of electrolyte Maximum discharge Discharge during normal operation Voltage/cell Self-discharge rate Topping up required Capital costs Suitability for photovoltaic use liquid 100% 20% 1.2 V high minimal high highly recommended Type of battery Lead–calcium antimony (2%) liquid 80% 20% 2V low infrequent mid-range highly recommended Lead–calcium antimony (6%) liquid 80% 20% 2V medium frequent mid-range recommended Lead–calcium liquid 50% 20% 2V low infrequent low not recommended

2. Setting up a peripheral health laboratory

15

Maintenance of lead batteries The daily discharge of lead batteries should not exceed 20% of the batteries’ capacity, otherwise the lifetime of the batteries (normally about 1100 recharge cycles), will be shortened. If the batteries are repeatedly discharged to 40% of their capacity, they will last for only about 600 cycles. (There are some special lead batteries available that can be discharged by 40%, but will last for about 3000 recharge cycles.) For maintenance the level of fluid must be checked regularly and when necessary refilled with the distilled water that is used for car batteries. High-efficiency batteries cannot be replaced by normal car batteries in case of a breakdown.When only car batteries are available to replace a defective high-efficiency battery, all the batteries in the energy storage system must be replaced with car batteries. Nickel–cadmium (Ni–Cd) batteries Ni–Cd batteries can be recharged by a solar panel. Some Ni–Cd batteries are the same size, but have different capacities. The AA-size Ni–Cd battery is available with a capacity from 0.5 Ah up to 0.7 Ah. Choose the batteries with the highest capacity. The small Ni–Cd batteries, type AAA to D, for use in laboratory instruments should be recharged in advance to enable continuous operation in a laboratory. The lifespan of Ni–Cd batteries may be 1000 recharging cycles, depending on their quality. Maintenance of Ni–Cd batteries Ni–Cd batteries appear to work unreliably in tropical countries. This apparent unreliability is caused by an increased rate of discharge rather than inefficient recharging of the battery at high ambient temperatures (see below). Such problems may be partially overcome as follows:
q

Ni–Cd batteries should be recharged at a low ambient temperature (e.g. in a refrigerator or in a specially constructed recharging box) shortly prior to being used. (For example, only 62% of the energy can be made available from a Ni–Cd battery that was charged at 40 °C.) Recharged Ni–Cd batteries should be stored under cool, dry conditions to minimize their rate of self-discharge. (For example, a Ni–Cd battery stored for 2 weeks at 40 °C will have a residual capacity of only 32%.) High humidity will also accelerate the self-discharge of the battery.

q

2.2.2 Setting up simple electrical equipment
If the laboratory has an electricity supply the following equipment can be used: — an electric lamp for the microscope (stable illumination makes adjustment easier); — an electric centrifuge (much faster than the manually operated type); — a microhaematocrit centrifuge (for detection of anaemia); — a spectrophotometer or colorimeter (allows accurate estimation of haemoglobin); — a water-bath, refrigerator etc. You may have to make simple connections or repairs to this equipment in the laboratory. The explanations given below are intended to help the laboratory technician to do this and are limited to the steps to follow in each case. Inexperienced persons should start by carrying out the procedures in the presence of an instructor.

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Manual of basic techniques for a health laboratory

The electricity meter (Fig. 2.3) An electricity meter measures and records the amount of electricity used. It indicates: — the voltage, measured in volts (220 V, 110 V, etc.); — the strength of the current, measured in amperes (A); — the frequency of the alternating current, e.g. 50 hertz (Hz) (cycles per second). Some types of meter have switches or buttons: — a flip-switch that can be flipped one way to cut off the electricity supply to the whole building (the mains fuse) and the other way to restore it; — a button marked “OFF” that can be pushed to cut off the electricity supply;
ON

— a button marked “ON” that can be pushed to restore the electricity supply. The flip-switch or “OFF” button also acts as a circuitbreaker, automatically cutting off the current when the circuit is overloaded. When this happens, first find and correct the fault that caused the cut-off, then press the “ON” button or flip the switch to restore the current.

OFF

Fig. 2.3 An electricity meter

Setting up new electrical equipment Voltage Check that the voltage marked on the instrument is the same as that of your electricity supply. The instrument has a label on it stating the voltage with which it must be used. The voltage of your electricity supply is marked on your electricity meter. Dual-voltage equipment Dual-voltage instruments can be used with two different voltage supplies. There is a device on the instrument that enables you to select the appropriate voltage, i.e. the voltage marked on your electricity meter. Depending on the instrument, this device may be: — a lever or switch that can be moved to the 110 V position or the 220 V position (Fig. 2.4(a)); — an unwired plug that can be transferred from the 110 V position to the 220 V position (Fig. 2.4(b)); — a screw that can be turned to the 110 V position or the 220 V position (Fig. 2.4(c)).
(c) (a) (b)

Fig. 2.4 Dual-voltage instruments

2. Setting up a peripheral health laboratory

17

The electrical power of the instrument The electrical power is measured in watts (W) and is marked on the plate that shows the correct voltage for the instrument. Each piece of electrical equipment in the laboratory uses a certain amount of power. The total power used at any one time must not exceed the power of your electricity supply. You can work out how much power is available from the figures shown on the meter: multiply the voltage (V) by the current (A). For example, if the voltage is 220 V and the current is 30 A, the electrical power supplied will be 220 ¥ 30 = 6600 watts or 6.6 kW. Using a transformer If an instrument is intended for use with a voltage different from that of the laboratory electricity supply, it can be used with a transformer. For example, if the centrifuge provided only works at 110 V and the voltage of your electricity supply is 220 V, ask for a 110 –220 V transformer, indicating the wattage of the centrifuge. Plug the centrifuge into the 110 V connection of the transformer supplied, then plug the 220 V lead from the transformer into the laboratory electricity supply (wall socket). Switching off electrical equipment After an instrument has been switched off, it must be unplugged from the wall socket. If left plugged in, it is a fire risk.

2.2.3 What to do in case of failure of electrical equipment
If an instrument does not work, check the following: — the fuses — the plug at the end of the cable — the cable — the wall socket — the voltage of the instrument and that of the electricity supply. Before doing anything, cut off the electricity supply: — either by pressing the button or the switch marked “OFF” on the meter — or by removing the mains fuse (Fig. 2.5). Tools (Fig. 2.6)
q q q q q

Screwdriver Wire-cutters Flat-nose or taper-nose pliers Fuse wire Various spare parts: plugs, switches, etc.

Fig. 2.5 Removing the mains fuse

Fig. 2.6 Tools for electrical work

18

Manual of basic techniques for a health laboratory

Changing the fuse Remove the cover from the fuse box. If it is a screw-type fuse, the fuse wire is stretched between two screws. If the wire is broken or melted, the current no longer passes: the fuse has blown. Loosen the two screws (Fig. 2.7). Remove the old fuse wire. Replace it with new fuse wire of the same gauge (thickness), or with thinner wire if the same size is not available. Fix the wire in an “S” shape, with a loop at either end. The wire must pass beneath the small washers under the screws. If it is a two-pin fuse, fix the fuse wire to the base of the pins, and then tighten the pins with pliers (Fig. 2.8). Once the fuse has been repaired, check the whole circuit before switching on the electricity supply. Checking the plug If a fault is suspected in a plug, it must be repaired or replaced. There are many different types of plug; some have a screw on the outside that can be unscrewed so that the cover can be removed.
Fig. 2.8 Changing a two-pin fuse

Fig. 2.7 Removing fuse wire from a blown fuse

Two-pin plug (Fig. 2.9) Inside the plug, the two wires of the cable are fixed to the terminal screws (T) of the contact pins (P). Check that the terminal screws are tightened. Sometimes this is all that is needed to repair the plug. Fitting a new plug To fit a new plug, remove the insulating material along a length of 1.0–1.5 cm from the end of each of the two wires making up the cable. This can be done by scraping with a knife but take care not to damage the wire inside. Twist the exposed ends of both wires to allow them to fit neatly into the terminal once the screw has been loosened (Fig. 2.10). Insert one exposed end into each of the terminals of the plug. Tighten the terminal screws and replace the terminals (Fig. 2.11). The screws should hold the wires firmly; check by pulling the wires gently.

Fig. 2.9 A two-pin plug

Fig. 2.10 Twist the exposed ends of both wires

Fig. 2.11 After inserting the wires into the plug terminals, tighten the terminal screws

Three-pin plug (Fig. 2.12) Two of the pins are connected to the electricity supply; one is “live” and one is “neutral”. The third (usually the middle) pin is connected to the “ground” or “earth”. It is most important to connect each of the three wires in the cable to the correct pin, and the plug usually contains instructions that should be strictly followed. If there is the slightest doubt, consult an electrician.

2. Setting up a peripheral health laboratory

19

Fig. 2.12 A three-pin plug

The ground or earth wire is covered in green or green and yellow insulating material. It provides an escape for the electric current in case of poor insulation, thus avoiding passage of the current through the human body. Checking the cable or switch Check to see whether the cable is burned or broken. If so, it should be replaced. There are many different types of switch. They have to be unscrewed and opened if you want to check that they are working properly. Make sure that the two incoming wires and the two outgoing wires are firmly fixed in their respective terminals (Fig. 2.13).

Fig. 2.13 A switch

Extension lead An extension lead is a cable with a male plug (M) on one end and a female plug (F) on the other (Fig. 2.14). The female plug is fixed to the cable by two terminals inside the plug, just as in the normal male plug. Checking the wall socket To check a wall socket, plug in a lamp that you know to be working. Some sockets are fitted with a small replaceable fuse. If this is not the case, it is usually wise to call in an electrician to repair a wall socket. Precautions
q

Fig. 2.14 An extension lead

Never take electrical equipment apart without first disconnecting the electricity supply. Never touch electrical equipment with wet hands (water is a good conductor of electricity).

q

etc.15 Tools and materials for plumbing repairs Important: Before starting any plumbing operation. W: washer. 2. H: head. there is a joint (J) of rubber or tow. Never replace fuse wire with wire that is thicker. Some simple remedies are described below. 2. cut off the water at the mains. Never remove a plug from a socket by pulling the cable. through which the water flows — the head (H). Between the head and the body.3.16 Components of a tap B: body. 2. a blocked sink.20 Manual of basic techniques for a health laboratory q Never plug a new piece of equipment into the electricity supply without first checking the plate to see whether the voltage marked is the same as that of the laboratory supply (110 V. etc. 2.15) q q q q q q Adjustable wrench Pipe wrench Set of screwdrivers Bottle brush Rubber washers for taps Rubber stoppers such as those used in penicillin bottles Plunger for clearing blocked pipes Tow and jointing compound for sealing joints. 2. Fig.17 Removing the head of a tap . J: joint. 2. 220 V.). 2.1 Tools and materials (Fig.3 Plumbing: simple procedures A fault in the plumbing of the laboratory (a dripping tap.3. in case a plumber is not readily available.2 Taps A tap is made up of two parts (Fig.16): — the body (B).) can hamper laboratory work considerably. if available. q q 2. which controls the flow of water by means of a rubber washer (W). q q Fig. Fig.

Wind new tow around the screw thread. 4. This will act as a temporary seal until a plumber can be called in. What to do if water leaks out of the head of the tap If water leaks out of the head of the tap. the joint needs to be replaced.21). 2.20). 3. pull it out.2. Unscrew the head of the tap using an adjustable wrench (turn in an anticlockwise direction) (Fig. 3. 2. Unscrew the head of the tap using an adjustable wrench.18(a)). Fig. 1. the body ends in a large screw (S) (Fig. unscrew it. starting at the top and winding in a clockwise direction (Fig. Smear jointing compound over the tow (Fig. 2. Replace it with a new washer of the same type.19 Repairing the seating for the washer S: seating. If the joint is made of tow: 1. 2.18 Removing the washer B: base of the head of the tap. 2. 2. 2.20 Removing the tow from around the screw thread . 2.19(b)). 2. Replace the joint with a new one of the same type. Setting up a peripheral health laboratory 21 What to do if water flows when the tap is turned off If water continues to flow when the tap is turned off. In this case place a rubber stopper in the hole (Fig. 2. Replacing the whole tap Unscrew the faulty tap. 4.18(b)).17). Remove the old joint. 2.19(a)) is probably faulty. scraping the screw thread with a pointed knife (Fig. If it is screwed on (Fig. 2. the washer needs to be replaced. 2. the seating (S) that receives the washer (Fig. Wind tow around the thread and smear with jointing compound as described above. Fig.23(a)).22). Take the new tap. 1. If the washer is embedded (Fig. Replace the head of the tap on the body and screw down as far as it will go. using a pipe wrench (turn in an anticlockwise direction). Remove the worn washer from the base of the head (B). (a) (b) Fig. 2. If the tap continues to leak after the washer has been replaced. 2.

2. J2.24) The sink trap consists of: — the body. The wastewater flows into the trap. Tighten with the wrench. Screw the new tap into the water pipe in the wall in place of the old one (Fig. The whole trap is attached to the waste pipe by a joint ( J3). 2.23(b)).3 Sink traps Components of a sink trap (Fig. This prevents foul air from the waste pipes and sewers from coming up into the sink. which is permanently filled with water (the seal).23 Replacing a tap S: screw. J3: joints. Fig. fixed to the body by a joint ( J2). 2.3. 2. . fixed to the sink outflow by a joint ( J1). 2.22 Applying jointing compound to the tow Fig. — the swan neck of the U-shaped trap.24 Components of a sink trap J1. 2.21 Winding new tow around the screw Fig. 2.22 Manual of basic techniques for a health laboratory Fig. Sink traps may become blocked so that wastewater from the sink or basin cannot drain away.

the permanent reservoir of water (the seal) at the bottom of the trap must have leaked because of a fault in joint J2. Press down on the wooden handle to flatten the plunger (Fig. over the waste pipe. It requires: — clean water — distilled water — demineralized water (if possible) — buffered water (if possible). Heat the components in diluted acetic acid (20 ml of acid per litre of water). 2. 2. 2. Repeat this procedure several times. as fast as you can.25). Unblocking with chemicals Use a commercial product intended for the purpose.26). What to do if the sink trap is leaking If foul smells come up through the waste pipe of the sink. Screw the joint down tightly. Setting up a peripheral health laboratory 23 Unblocking with a plunger Place the plunger over the waste pipe. If it is splashed on the skin or in the eyes. Warning: Sodium hydroxide solution is highly corrosive and should be used with extreme care. Leave for 5 minutes. Unblocking by emptying the sink trap Place a bucket beneath the trap. The suction caused may break up whatever is blocking the sink.27 Replacing the seal at the bottom of a sink 2. wash the affected areas immediately with large quantities of water. Unscrew joint J2 using an adjustable spanner (Fig. Let a little water flow around it to help it stick. . Clear away all waste material. Important: Never pour strong acids down a sink. then rinse the sink thoroughly with cold water from the tap.27). 2. Pour 2 litres of boiling water on to the pellets (avoid splashing).4 Water for laboratory use The medical laboratory needs an adequate water supply for its work. AlFig.2. take it apart completely. Reassemble the sink trap. use 250 g of sodium hydroxide pellets. If there is a white deposit (limescale) in the trap. 2. Pull it up and then push it down hard again. Clean the trap with a bottle brush or piece of wire.25 Unblocking a sink with a plunger ternatively. or replace it with a new one (Fig. Put the pellets in the bottom of the sink or basin. since they can cause corrosion.26 Unblocking a sink by emptying the sink trap Fig. Fig. 2.

the water needs to be filtered. Fig.28). Fig.g. Storage of water If water is scarce or comes from a tank or well.4.30): 1. a big earthenware pot or a perforated bucket) — sand (S) — gravel (G). 2. You will need the following (see Fig. Preparation Distilled water is prepared using a still. Attach a length of rubber tubing to the container so that the water can flow down.1 Clean water To check whether the water supply is clean. 2. Using a sand filter A sand filter can be made in the laboratory. it can be kept immersed in a container of the water to be filtered (Fig. 2. minerals) but it may contain volatile organic compounds. Decant water that has been stored before filtering it. in which ordinary water is heated to boiling point.29 Filtering water using a sand filter G: gravel. Important: Filters of this type must be dismantled once a month and washed in boiling filtered water. 2. S: sand. If there is a deposit. Clamp the rubber tubing with a Mohr clip or a small screw clamp. Water supply If there is no running water in the laboratory. 2.24 Manual of basic techniques for a health laboratory 2.29): — a filter reservoir (a large container such as a metal drum. Fig. 2. and the steam produced is cooled as it passes through a cooling tube where it condenses to form distilled water.30 A water distributor . fill a bottle with water and let it stand for 3 hours. 2. Place the container of water on a high shelf. but it may contain water-soluble chemical compounds and bacteria. Examine the bottom of the bottle. set up a distributor as follows (see Fig.28 Filtering water using a porous unglazed porcelain or sintered glass filter 3. 2. preferably in glass or plastic containers. Alternatively.2 Distilled water Distilled water is free from nonvolatile compounds (e. always keep a large supply in reserve. Filtering Using a porous unglazed porcelain or sintered glass filter This type of filter can be attached to a tap.4. Note: Water that has been filtered through a sand filter is almost free of particles.

2. Setting up a peripheral health laboratory

25

The following types of still are available: — copper or stainless steel stills (alembics) — glass stills — solar stills. They are heated by gas, kerosene, electricity or solar energy, depending on the type of still. Copper or stainless steel alembics (Fig. 2.31) 1. Fill the reservoir (R) with the water to be distilled. 2. Connect the cold-water tube (T) to a tap. 3. Heat the reservoir with a Bunsen burner (B) or kerosene heater. The still can produce 1 or 2 litres of distilled water per hour, depending on the efficiency of the heating system.

Fig. 2.31 Components of a copper or stainless steel alembic B: Bunsen burner; C: cooling column; R: reservoir; T: cold-water tube.

Glass stills (Fig. 2.32) Glass stills are more fragile, but almost always produce purer water than metal stills. The distillation method is the same. Make sure that the running water circulates freely round the condenser (C). The water can be heated in the flask by the electric element (E).

26

Manual of basic techniques for a health laboratory

Fig. 2.32 Components of a glass still C: condenser; D: distillate; E: electric element.

Fig. 2.33 Components of a solar still

Solar stills (Fig. 2.33) For laboratories in remote areas and with limited resources, a simple solarpowered water still can be easily constructed using a clean plastic container with two compartments (one large and one small) and a large surface area, over which is placed a glass cover in a sloping position. The water is poured into the large compartment from which it is evaporated by the sun. It condenses on the glass cover and drops into the small compartment. The small compartment has an outlet at the bottom through which the distilled water can pass into a glass bottle placed underneath the container.

2. Setting up a peripheral health laboratory

27

In tropical climates 2–7 litres of distilled water can be produced daily from a solar still with a surface area of 1 m2. Important:
q q

Collect the distilled water in a glass or plastic container. Do not distil the last quarter of the water heated; it contains residues.

Quality control The pH of distilled water is normally between 5.0 and 5.5 (i.e. it is acid). Use a 1.7% solution of silver nitrate (AgNO3) (reagent no. 49) to check for the absence of chloride compounds (e.g. calcium chloride). Put in a beaker: — 10 ml of distilled water; — 2 drops of nitric acid; — 1 ml of silver nitrate solution. The water should remain perfectly clear. If a slight whitish turbidity appears, the distillation process should be repeated. Uses Distilled water is used for the preparation of reagents and as a final rinse for some glassware before drying. Important:
q

Do not use commercial distilled water (the type sold for filling car batteries) for the preparation of laboratory reagents. Freshly prepared distilled water is preferable; if this is not available, use distilled water stored in glass or plastic containers, which should be washed periodically. Always use distilled water prepared the same week.

q

q

2.4.3 Demineralized water
Principle Demineralized water is free from ions but not necessarily free from organic compounds. Preparation Demineralized water is prepared by passing ordinary water through a column of ion-exchange resin. The apparatus consists of a long cartridge filled with ionexchange resin granules. The water filters through the column of granules, which retain all the mineral ions (i.e. all the dissolved mineral salts). Some demineralizers have two cartridges through which the water passes successively (Fig. 2.34). 1. Check that the cartridge is completely filled with ion-exchange resin granules. 2. Connect the inlet tube of the apparatus to the water supply (a tap or a small tank placed above the apparatus). In some models the water flows in at the top of the column, in others it flows in at the bottom. 3. Let the water flow in slowly. 4. Collect the demineralized water in a closed container.

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Manual of basic techniques for a health laboratory

Fig. 2.34 A demineralizer

Quality control Apparatus with a control dial The dial registers the resistivity of the water resulting from the presence of ions. The more complete the demineralization, the higher the electrical resistivity of the water. 1. Check that the control system is fitted with a battery in good working order. 2. To check that the battery is charged, press the button marked “zero test”; the needle on the dial should swing to zero (Fig. 2.35(a)). 3. Let water flow into the cartridge. 4. When demineralized water begins to flow out at the other end, press the button marked “water test”. The needle should register a resistivity of over 2 megaohms/ cm (2 MW/cm) (Fig. 2.35(b)). 5. If the needle stops at a point below 2 MW/cm or stays at zero, the cartridge of ion-exchange resin granules has been used for too long and must be replaced or reactivated. The apparatus may indicate the resistivity (MW/cm) or the reciprocal value, the conductivity (cm/MW or Siemens, S). Apparatus without a control dial Using an indicator paper, determine: — the pH of the water supply flowing into the apparatus, and — the pH of the demineralized water that flows out at the other end. If the pH remains the same (usually below 6.5), the resin is no longer active. Demineralized water should have a pH between 6.6 and 7.0. An additional check can be made using a 1.7% solution of silver nitrate (reagent no. 49). Pass a weak solution of sodium chloride (cooking salt) through the resin, then carry out the test described in section 2.4.2 for the quality control of distilled water. If a slight whitish cloudiness appears, the resin must be replaced.

2. Setting up a peripheral health laboratory

29

(a)

2

1

0.3 0.5

0.1

0

4

0

M /cm

(b)

2

1

0.3 0.5

0.1

0

4

M /cm

WHO 01.203

Fig. 2.35 Measuring the resistivity of demineralized water

Change of colour in resin If the resin changes colour (e.g. it turns black), consult the instructions for use supplied by the manufacturer. It may need to be reactivated or replaced, as described below. Replacement or reactivation of ion-exchange resin This can be done in one of the following ways, depending on the model:
q q

The cartridge is replaced by another filled with ion-exchange resin granules. The column of the apparatus is refilled with ion-exchange resin or a mixture of two resins. The exhausted ion-exchange resin is reactivated by passing a solution of ammonia through the apparatus. Follow the instructions supplied by the manufacturer.

q

Uses Demineralized water can be used for: — rinsing glassware before drying; — preparing almost all the reagents used in medical laboratories, including stains.

2.4.4 Buffered water
Distilled water is usually acid and demineralized water becomes acid on exposure to the air. For a number of laboratory procedures (preparation of stains, etc.) the

30

Manual of basic techniques for a health laboratory

pH of the water has to be around 7.0 (neutral water) and has to be kept neutral. This is achieved, if possible, by dissolving buffer salts in the water (buffered water). Materials and reagents
q q q q

Measuring cylinders, 10 ml and 1000 ml Volumetric flask, 1000 ml Universal indicator paper (for measuring pH from 1 to 10) Indicator paper of limited pH range: for the 5.0 –7.0 range and for the 6.0 – 8.0 range Distilled (or demineralized) water Acetic acid, 5% solution (reagent no. 1), diluted 1:10 with distilled water Disodium hydrogen phosphate (Na2HPO4·2H2O), hydrated Phenol red, 1% solution (reagent no. 42) Potassium dihydrogen phosphate (KH2PO4), anhydrous Sodium carbonate, 0.2% solution (reagent no. 51).

q q q q q q

Method 1. Weigh out accurately 3.76 g of disodium hydrogen phosphate. 2. Transfer the chemical to a 1000-ml volumetric flask through a funnel (Fig. 2.36). 3. Rinse out the weighing container into the volumetric flask several times with water. Rinse the funnel into the flask. 4. Weigh out accurately 2.1 g of potassium dihydrogen phosphate and proceed as in steps 2 and 3. 5. Add a little more water and mix the solution until the chemicals are dissolved. 6. Fill the flask to the 1000-ml mark with water. 7. Replace the flask stopper and mix the solution well. 8. Store the solution in a white glass reagent bottle and keep in a refrigerator.

Fig. 2.36 Transferring disodium hydrogen phosphate into a volumetric flask

2. Setting up a peripheral health laboratory

31

9. Dip a strip of the universal indicator paper into the buffer solution and compare the colour obtained with that shown on the standard chart (Fig. 2.37). Read off the pH unit given for the colour that matches the test paper most closely. 10. According to the result obtained, select a strip of indicator paper for the corresponding limited range. For example, if the pH is 6, use indicator paper for the range 5.0 –7.0. If the pH is 7.5, use indicator paper for the range 6.0–8.0. 11. Repeat the test, using the paper for the corresponding limited range. Read off the pH of the buffer solution on the standard chart.

Fig. 2.37 Checking the pH using universal indicator paper

12. If the pH is between 7.0 and 7.2, the buffered water is satisfactory. If it is below 7.0, the water is acidic. If the water is acidic, make a fresh solution, using distilled water that has been boiled for 10 minutes in an uncovered round flask (this gets rid of the carbon dioxide). 13. If the water is still acidic after boiling: — add five drops of phenol red solution for every litre of water; — neutralize by adding sodium carbonate solution, one drop at a time, until the water turns pink (Fig. 2.38). 14. If the water is alkaline (pH above 7.2): — add five drops of phenol red solution for every litre of water; — neutralize by adding acetic acid solution, one drop at a time, until the water turns orange (Fig. 2.39).

Fig. 2.38 Correcting the pH of acidic buffered water

Fig. 2.39 Correcting the pH of alkaline buffered water

If neither disodium hydrogen phosphate nor potassium dihydrogen phosphate is available, neutralize distilled or demineralized water directly, as shown in steps 12–14 above. Note: The pH can also be corrected by adding small quantities of the buffer salts:
q

Disodium hydrogen phosphate can be used to increase the pH if the water is acidic (pH below 7.0). Potassium dihydrogen phosphate can be added to reduce the pH if the water is alkaline (pH above 7.2).

q

1 2 3 For further information. bacteriology. q At the health centre level one binocular microscope is sufficient. ¥ 40. see section 3.32 Manual of basic techniques for a health laboratory 2. see section 3. Photometer or colorimeter It is necessary to have a photometer or colorimeter for blood chemistry tests and for accurate determination of haemoglobin levels.5 Equipment The following is a list of the apparatus needed to equip a laboratory capable of carrying out all the examinations described in this manual. If the laboratory is required to prepare a wide range of reagents.3.2.1. ¥ 100). a condenser and an electric lamp that can be connected to the mains electricity supply or a battery. a two-pan balance with a corresponding set of weights (see section 3. Balance3 An analytical balance with a set of weights is necessary if reagents are to be prepared in the laboratory. 2. see section 3. etc. specimens. two eyepieces (¥ 5. . For further information.5.1 Essential laboratory instruments Microscopes1 The laboratory should be equipped with two microscopes. It should have an inclined binocular tube. urine analysis. Refrigerator Reagents (such as those required for pregnancy tests.3. — a hand-operated or an electric centrifuge with four buckets.2) is useful.) and materials (such as certain transport media.) should be kept in the refrigerator. The second microscope is for use in other laboratory procedures (parasitology. Centrifuges2 It is useful to have two centrifuges: — an electric centrifuge with a microhaematocrit head attachment and a reader. Battery-powered models are commercially available. a mechanical stage. Differential counter Although a hand tally counter can be used. etc. Such a laboratory would usually be located in a small rural hospital (district level) which might have between 60 and 100 beds. ¥ 10). three objectives (¥ 10.) and should have an inclined binocular tube and accessories as listed above. etc. For further information. q One microscope is for use in haematology. a differential counter saves time. Water-bath A water-bath equipped with a thermostat for temperature control is required when samples or materials must be kept at a certain temperature and when measurements must be made at a given temperature.2.

If a deionizer is not available.5. Setting up a peripheral health laboratory 33 2. Materials q q q q q Hollow glass tubing with an external diameter of 4–8 mm and 0.2.5): — a small autoclave (electric or heated by an oil stove or with butane gas) — a pressure cooker. 2.3 Equipment and supplies A list of equipment and supplies for a peripheral-level health laboratory is given in Table 2. which is less brittle and more resistant to heat than ordinary glass.41).2. Using the file.5. mark off the required lengths of tubing: — 14–15 cm for small pipettes. 2. a water still can be used (see page 25).4 Making glass equipment Glass is produced by the fusion at a very high temperature of sand and potassium (or sodium).5).40. Making a Pasteur pipette 1.2 Additional items Autoclave If the laboratory is in a hospital.5.5. — 18–25 cm for large pipettes. Deionizer or water still A deionizer is an apparatus for demineralizing water by means of cartridges filled with ion-exchange resin (see section 2. 2. forming a circle (Fig. the hospital sterilization service can be used. The quantities proposed are sufficient to enable a laboratory with one or two technicians to perform 20–50 examinations per day for a period of 6 months. . glass cutter or diamond pencil Cloth Bunsen burner (or a small gas or petrol blowlamp).3).9–1. This forms a silicate (ordinary soda-lime glass). If the laboratory is in a health centre one of the following is needed (see section 3. Etch the mark right round the tube.0 mm thick Glass rods with a diameter of 4–8 mm File. Hot-air oven If the laboratory is fairly large.5. Certain pieces of equipment can be made in the medical laboratory by heating ordinary glass. Sometimes boric acid is added to the ingredients to produce borosilicate glass.4. Take a piece of glass tubing 4–6 mm in diameter. Glassware and small items of equipment for laboratory use are shown in Fig. 2. a small hot-air oven is useful for drying glassware and for sterilization in conjunction with the autoclave (see section 3.

2.40 Glassware and equipment for laboratory use .34 Manual of basic techniques for a health laboratory (a) Fig.

2. Setting up a peripheral health laboratory 35 (b) .

100 ml Measuring cylinders. 25 ml. plastic or cardboard.1 mm) ¥ 40 mm Needles. graduated. disposable. for taking skin biopsies (for onchocerciasis) Tongue depressors. 5 ml Needles. with metal screw cap and rubber washer. punch.5 ml and 5 ml. graduated.9 mm) ¥ 40 mm Needles. 250 ml 3 4 4 4 4 1 2 1 3 3 3 2 1 1 3 1 1 50 50 50 25 25 20–40 1 50 as needed as needed as needed as needed as needed as needed as needed as needed as needed 2 pieces as needed 2 ¥ 500 g 2 ¥ 500 g as many as possible Quantity required . reagents. absorbent Cotton wool. glass. glass. 2–5 mm bore Lancets for taking capillary blood Cotton wool. disposable. graduated. disposable. white glass. glass.2 mm) ¥ 40 mm Needles.0–1. for stool collection Applicators. plastic. 23-gauge (0.7 mm) ¥ 40 mm Needles. 19-gauge (1. wide-mouthed. Erlenmeyer. disposable. 20 ml) Additional equipment Scalpel with disposable blades for taking slit skin smear specimens (for leprosy) Curved clamp forceps without teeth for taking slit skin smear specimens (for leprosy) Boxes. preferably plastic Bottles. for injection (5. flat. glass. 250 ml Staining troughs. wooden (12 cm ¥ 1 mm) (can be made locally) Bottles.6 mm) ¥ 32 mm Needles. graduated. non-absorbent Bottles. disposable. 6 mm diameter Beakers. all sizes. wooden Glassware Essential items Glass rods. 20 ml Syringes. wide-mouthed. graduated. etc. white. wide-mouthed. with metal screw cap and rubber washer. 50 ml. glass. graduated. 100 ml Beakers. heat-resistant. plastic. for collection of sputum specimens Bottles. disposable. disposable.2 Equipment and supplies for a peripheral-level health laboratory Item Equipment for collection of specimens Essential equipment Syringes. 18-gauge (1. 50 ml Measuring cylinders. white glass.36 Manual of basic techniques for a health laboratory Table 2. 60 mm diameter Funnels. 10 ml Syringes. glass. 90 mm diameter Funnel. 23-gauge (0. plastic. 500 ml Measuring cylinder. graduated.6 mm) ¥ 90 mm Rubber tubing for tourniquet. 20-gauge (0. 1000 ml Flasks. 50 ml Beakers. disposable. for collection of urine specimens Forceps. flat. disposable. for 20 slides Funnel. graduated. 2. glass. 250 ml Measuring cylinder. 22-gauge (0. rectangular. 25 ml Measuring cylinders. plastic. disposable. previously containing antibiotics. glass. flat. 200 mm diameter Measuring cylinders. 10. graduated. for various specimens Bottles. solid.

optically plane. 112 mm diameter Petri dishes. heat-resistant. with stoppers. Setting up a peripheral health laboratory 37 Table 2. Pasteur Test-tubes. 500 ml Flasks. 100 ml Reagent bottles. 1000 ml Flasks. 0. 500 ml Flask. 2 ml (0. 75 mm diameter (75 ml) Dessicator Equipment for haematology tests Pipettes. 5 ml (0. volumetric. Westergren. 250 ml Flasks.1-ml subdivisions) Glass tubing. graduated. 1000 ml Watch glasses. 25 mm ¥ 75 mm Coverslips. heat-resistant. 15 ml Centrifuge tubes.5 mm thick. graduated from the top (and not to the tip). plastic or glass. heparinized Wax.05 ml Counting chambers. glass. 0. graduated from the top (and not to the tip). for determination of erythrocyte sedimentation rate Stands for Westergren tubes Microhaematocrit centrifuge Microhaematocrit capillary tubes. Sahli.0–1. conical. 50 mm diameter Pipettes. for counting chambers Tally counter Tubes. plastic or glass.01-ml subdivisions) Pipettes. heat-resistant. with stoppers. Fuchs–Rosenthal Coverslips. 500 ml Wash bottles. 7–8 mm diameter Additional items Petri dishes. plastic or glass. volumetric.01-ml subdivisions) Pipettes. for sealing microhaematocrit tubes Equipment for bacteriological and biochemical tests Nichrome wire. volumetric. wide-mouthed. 100 ml Drop bottles. glass. volumetric. 50 mm ¥ 6 mm (cross-matching tubes) Centrifuge tubes.2 (cont.02 ml.1-ml subdivisions) Pipettes. conical. 20 mm ¥ 20 mm Wash bottles. 156 mm diameter Evaporating dishes. 85 mm ¥ 15 mm (Kahn tubes) Test-tubes. heat-resistant. 1 mm diameter 1m 30 20 3 1 12 1 30 2 1 1000 1 roll 4 4 2 1 Quantity required 3 3 12 3 20 10 10 4 2 2 1 2 ¥ 1000 20 ¥ 100 2 2 2 12 10 10 6 2 ¥ 144 50 100 20 40 50 1 kg . with rubber tubing Pipettes.) Item Flasks. heat-resistant. 1. 100 ml Flasks. plastic or glass. Erlenmeyer. graduated from the top (and not to the tip). glass. plastic. 15 ml (0. 500 ml Reagent bottles. 1000 ml Drop bottles. 1000 ml Microscope slides. improved Neubauer (bright line if possible) Counting chamber. graduated from the top (and not to the tip). 10 ml (0.1-ml subdivisions) Pipettes. wide-mouthed. 150 mm ¥ 16 mm Test-tubes. glass. plastic.2. with stoppers. 1 ml (0. with stoppers. 100 ml Reagent bottles. glass. blood. glass. Erlenmeyer. brown glass.

0–60 min. lead Pens. blue Glass marker. for 12 tubes Wooden test-tube holders Forceps. ballpoint. red ink (for recording positive specimens) Pens. white Labels for specimen bottles Laboratory request forms (preferably standardized centrally) Miscellaneous equipment Microscopes Colorimeter Water bath Refrigerator Hot-air oven Centrifuge Balance Deionizer or water still Timer. diamond point Pencils. hardbacked. 50 cm ¥ 30 cm Bucket. large. 5 mm Small ampoule files Saucepan.2 (cont. for 12 tubes Test-tube racks. small. large Round metal file. 20 ml 2 1 1 1 1 1 2 1 1 1 1 1 pair 1 pair 1 1 1 1 12 1 1 1 3 1 4 1 6 12 12 1 12 2 3 3 rolls 3 rolls 1000 as needed Quantity required 4 1 1 set 4 4 2 2 1 as needed 1 3 . 12 litres Rubber safety bulb Micropipette. ballpoint.38 Manual of basic techniques for a health laboratory Table 2. wax. plastic. medium Screwdriver. flat-bottomed with lid. for weighing reagents Laboratory records and reports Record books. with alarm Spirit lamp Hammer Pliers Pliers. various sizes. small Screwdriver.) Item Loop holders Wooden block for loop holders Protein standard tubes Test-tube racks. black or blue ink Cellophane tape Adhesive tape. large Glass-marking pencils. plastic. 30 cm diameter Hot plate Pestle (10 cm diameter) and mortar Bowls. electrician’s Screwdriver. wax. red Glass-marking pencils. for slides Bunsen burner for use with butane gas Butane gas cylinder Tripod with asbestos gauze Spatulas. stainless steel.

42 Breaking the glass tubing by hand Fig. 100 ml Micropipette tips. Round off the end of each piece of tubing as shown in Fig. narrow range (6. disposable. 15 cm diameter (Whatman’s No.2) pH paper. Snap by pressing with your thumbs. disposable. holding the tube almost vertical just above the blue flame of the burner. 1 or equivalent) pH paper. plastic. rubber Stoppers. 2.2 (cont. large Vacuum pump. 50 ml Micropipette. 2.) Item Micropipette. 20 ml Micropipette tips. 2. disposable.41 Marking off the required length of glass tubing using a file Fig.8–7. 2. 200 ml Micropipette tips. 200 ml Micropipette. 2. 500 ml Scissors. plastic. disposable. 100 ml Micropipette. medium Scissors. 50 ml Micropipette tips. soft camel-hair brush or blower (for cleaning lenses) Small rubber bulb (for cleaning lenses) Toilet tissue Towels and clean rags Immersion oil Quantity required 1 1 1 1 as needed as needed as needed as needed as needed 1 1 1 1 1 set 1 set 1 6 4 boxes 6 books 6 books 2 packets 1 1 10 rolls as needed 6 bottles (10 ml each) Fig. 0–100 °C Stoppers. plastic. 3. plastic. one thumb on either side of the etched mark (Fig.43: — heat the end. metal Thermometer. disposable. Wrap the part to be broken in a cloth. Hold the tube with both hands. Setting up a peripheral health laboratory 39 Table 2.2. . 500 ml Micropipette tips. wide range (0–12) Lens paper Fine paintbrush.42). plastic.43 Rounding off the ends of the glass tubing by flaming 2. cork Corkscrew Test-tube and bottle cleaning brushes (various sizes) Filter-paper.

2. 4. 2. 2. Rinse and dry. Fig. Round off the ends by rotating them over the blue flame of the burner. Fig.52).41).1). still rotating it continuously. Alternatively. 2.47 Rounding off the ends of the glass rod by flaming .44 Heating the glass tubing before pulling the pipette — keep rotating the tubing until the glass becomes red. and leave to cool. 2. Use a glass rod about 5 mm in diameter. Cut off the drawn portion at the exact length required.44). 2. until about 1 cm of the rod is bright red (Fig. Pull the glass to the length required (10–20 cm). Glass rods can be used to decant liquids or to pour them slowly (see Fig.45 Pulling the pipette Fig. Remove the tubing from the flame. Pulling the pipette is carried out as follows: — heat the middle of the length of tubing over the blue flame (Fig. heated ends up. Making a stirring rod 1. 20 or 25 cm according to requirements.5. using a file (see Fig. 2.46).47). Cut the rod into lengths of 15. 4.45). 2.49). Leave to cool. and pull the two ends apart slowly. keeping your hands perfectly level (Fig.46 Rounding off the ends of the pipette by flaming 6. Heat the other end and press it gently down on the tiled surface (Fig. Round off the sharp edges by holding them for a few seconds in the flame (Fig. Wash all the pieces of tubing prepared (following the instructions given in section 3. — stop when the glass becomes red hot. 2. 3. Fig. 2. 5. Flatten the heated end against the (dry) tiled working surface with a 500-g or 1-kg weight (Fig. separate and seal the two pipettes by heating the pulled-out portion in the flame. 2. Stand the tubes in a beaker or a can. 7. 2.48). 3.40 Manual of basic techniques for a health laboratory — keep turning slowly.

51 Bending glass tubing to make a right angle Bending glass tubing 1. 2. 2.49 Pressing the heated end of the glass rod down on a tiled surface Fig. rotating the tubing over the flame until the glass turns pale red (Fig.51). Fig. 1000 ml Two pieces of glass tubing Cork or rubber stopper. 2. Heat the spot where the bend is to be made. Making a wash bottle Materials q q q Erlenmeyer flask.2. 2. 2. 2. 2. 2.50) and sags. Setting up a peripheral health laboratory 41 Fig. Poor bends (Fig.52 Common problems with bending glass tubing . Fig.48 Flattening the heated end of the glass rod using a weight Fig.52) Poor bends may result if: — the glass was too hot (a) — the glass was not hot enough (b).50 Heating glass tubing before bending Fig. Bend the heated tubing slowly to make a right angle (follow the corner of a tile. 2.

2.55) (or use 0. also known as edetic acid.5. urine and sputum in the laboratory. Moisten the ends of the tubing with a few drops of water (for cork) or glycerol (for rubber) before inserting them in the holes (Fig. blood. 2. Trisodium citrate Trisodium citrate. 1 Ethylene diamine tetraacetic acid. 2. Bottles and test-tubes for collecting blood specimens Without anticoagulant The best type of test-tube to use for blood specimens is one that can be centrifuged: this avoids excessive handling of the specimen. q Use clean dr y test-tubes of 5–20 ml capacity. Heparinized tubes Heparin is an expensive anticoagulant that is not very stable in hot climates.53 Components of a wash bottle — empty tin with a lid — light plastic box — glass jar specially designed for stool collection.2 ml in 2-ml bottles). depending on requirements. 22) into each of a series of 5-ml bottles (Fig.5 ml of EDTA dipotassium salt. with a spoon attached to the stopper. 2. 3. Containers for stool specimens The following types of container are suitable for the collection of stool specimens (Fig.54 Containers for stool specimens Use these bottles for: — blood cell counts — haemoglobin estimation.5 Specimen containers Different types of containers are used for the collection of specimens such as stools. Place the open bottles in an incubator at 37 °C or leave them to dry at room temperature.54): — waxed cardboard box Fig. Protect your hands with a cloth. With anticoagulant for haematological tests EDTA1 dipotassium salt Put 0. 2. Use 1 ml of trisodium citrate solution per 4 ml of blood (or 0.4 ml per 1.42 Manual of basic techniques for a health laboratory Method Pierce the stopper with a cork borer. 10% solution (reagent no. 2. Heparinized tubes are usually obtained commercially or prepared by central laboratories and are already marked to show the level to which the blood should be added. 60) is used for the determination of the erythrocyte sedimentation rate.6 ml of blood).2% solution (reagent no. Fig. . if no incubator is available.53).

Warning: Traces of detergent will dissolve the erythrocytes. 5 ml. or 2 mg per 2 ml of blood. Use bottles and tubes with a graduation mark. Setting up a peripheral health laboratory 43 Fig. or stick on a label so that its upper edge corresponds to the required amount of blood (2 ml. Do not shake. With anticoagulant for biochemical tests Sodium fluoride (NaF) is the anticoagulant normally used for biochemical tests. Precautions to be taken when using anticoagulants q Mix as soon as the blood is collected by inverting the bottle several times gently and evenly. etc.). 2. q q Store bottles containing anticoagulants in a dry place. Warning: Sodium fluoride is a poison. Use for: — blood glucose estimation — blood urea estimation (certain techniques). EDTA dipotassium salt solution and sodium fluoride are stable at room temperature but trisodium citrate solution and heparin must be kept in the refrigerator. Use 10 mg of sodium fluoride powder per 10 ml of blood. q .55 Dispensing EDTA solution into bottles for collection of blood specimens Important: Never carry out a blood cell count on citrated blood. Use the correct proportions.2. Dry before adding anticoagulant. Ensure that the bottles are rinsed thoroughly before drying. Use clean bottles.

58). Fold two of the corners back against one side.6. 2. Burn these cartons and plastic jars after use. and the other two against the other side (Fig. 3.59). q Boxes and jars for collecting sputum specimens Glass screw-top jars or disposable plastic jars with lids can be used for collecting sputum specimens. dry. These cartons can be used once only for sputum collected in the laboratory. Cerebrospinal fluid (CSF) — use test-tubes measuring 150 mm ¥ 16 mm. 4.56 Folding cardboard to make cartons for sputum collection Fig. 2. 2. 2. 2. See section 8. 2. wide-mouthed Erlenmeyer flasks of 250-ml capacity or clean wide-mouthed bottles. Cut out pieces of thin cardboard 18 cm square and fold them as shown in Fig. Fig.56: — first from corner to corner — then into nine equal squares. Fold the diagonal creases in each corner square inwards (Fig.57 Folding the corners inwards Fig. 5. 2. Staple the two folded corners on each side of the box (Fig.59 Securing the folded corners .2. 2.58 Folding two of the corners back against one side of the carton Fig.44 Manual of basic techniques for a health laboratory Bottles and tubes for collecting other specimens q Urine — use clean.2. 1. or small cartons can be made in the laboratory using cardboard and a stapler.57). which is now ready to receive the specimen. as described in section 3. 2.

12.g.6 Storage. Setting up a peripheral health laboratory 45 2.3. When you order an item.01 2 bottles 1 bottle 4 bottles 3 bottles 2 bottles 4 bottles Item no. indicate: — in the column headed “Issued”: the date of issue and the amount issued — in the column headed “In stock”: the total left in stock after the item has been issued.01 1 bottle 3.01 2 bottles 10. piece of glassware.: 29 In stock . Acids and inflammable and dangerous chemicals (indicated by appropriately coloured labels) should be stored separately in a special section. should be kept in an air-conditioned room if the climate is hot and humid. indicate: — in the column headed “Received”: the date of receipt and the quantity received — in the column headed “In stock”: the total in stock in the laboratory after the item has been received. stocktaking and ordering supplies Storage Glassware Keep glassware on the shelves of a cupboard away from dust. etc.01 2 bottles 20. Instruments Some instruments. Graduated pipettes should be kept in drawers divided into sections. Stocktaking Stock cards A stock card should be prepared for every chemical.11.8.01 Company A 15.01 2 bottles 10. indicate: — in the column headed “Ordered from”: where you sent the order — in the column headed “Ordered”: the date and the quantity ordered. When you receive an item.2. Table 2. stain. e. A sample stock card is shown in Table 2. When an item has been used up (or broken). spectrophotometers.6. For storage of microscopes see section 3.1. if available) and arranged by type and size. Chemicals and reagents Arrange chemicals and reagents in strict alphabetical order. Poisons (also indicated by appropriately coloured labels) should be stored separately in a locked cupboard.12. Unopened stocks can be kept in crates filled with sawdust. Erlenmeyer flasks should be plugged with non-absorbent cotton wool or covered with brown paper (or preferably with thin sheets of paraffin wax or clinging plastic.10.5.3 A sample stock card Item: Giemsa stain (250-ml bottle) Ordered from Date Ordered Quantity Date Received Quantity Date Issued Quantity 2 bottles Company A 1.8.

Inventory Make an inventory of all laboratory supplies every 6 months. March April Quantity used per month May June July Aug. This will: — prevent the specimens from getting mixed up.4 Estimating the quantity of supplies required Year Jan. — make the results available for the promotion of public health.1 Registration of specimens All specimens must be registered and given numbers when they arrive at the laboratory and the results of all investigations must be recorded. Expiry dates Reagents (e. Count the quantity of each item in stock and check that the figure corresponds to the one shown in the “In stock” column of the stock card. This expiry date should be marked on the container by the supplier. which is then entered on the stock card after the heading “Item no. Order two bottles every 3 months (or four bottles every 6 months if orders are submitted twice a year). etc. Each item can be given a number. For example: q q q Eight bottles of Giemsa stain have been used in a year. check the stock cards one by one. This gives an average of two bottles used every 3 months.6. Classify the stock cards in strictly alphabetical order and keep them in a box or filing drawer.4) is added to the bottom of each stock card. order the quantity used in a 3-month period. . It makes it easier to estimate the quantities required if a table summarizing the stock used each month (see Table 2. taking into account any recent increase or decrease in the amount used.g.6 Registration of specimens and preparation of monthly reports 2. Dec. stains and reagents. Sept. 2. To draw up the order.46 Manual of basic techniques for a health laboratory Table 2. In the case of chemicals. Ordering supplies A well-organized laboratory should submit an order to the central supply stores every 3 months. Oct. antigens. blood group antisera. — make it possible to look up a result. 2000 2001 2002 Feb.) have to be used before a certain date.”. Make a note of the expiry date on the stock card in the column headed “In stock”. Nov.

if there is none. . incorporate in the bacteriology register. The following registers are suggested: — haematology — blood chemistry — urine analysis — CSF examination — pregnancy tests — bacteriology — parasitology — mycology — serology (if the samples are few. 2. — monthly report forms. Laboratory registers Fig. and type of organisms. of leukocytes no. Write this number immediately: — on the request form — on the specimen container (use a grease pencil) — on every test-tube used for the specimen — on every microscope slide used for the specimen. 2. to the department of public health at both the provincial and the central level. This will prevent any mistakes. 2. For bacteriology: no. Setting up a peripheral health laboratory 47 The laboratory should have: — examination request forms that accompany the specimens. For example: q q For parasitology: no.6.2 Preparation of monthly reports At the end of every month the laboratory should submit a report to the director of laboratory services at the central level or.60) Give each specimen a number as soon as it is received. which should be modified according to your requirements. Numbering the specimens (Fig. otherwise keep a separate register) — histopathology — water analysis. of erythrocytes no. It is both helpful and time-saving to have rubber stamps for the most common tests and results. — a register for recording details concerning the specimens and the results obtained.2.60 Numbering the specimens Each numbered specimen should be recorded in a register for that type of specimen. of epithelial cells no. Tables 2. The report is valuable for two main reasons. of ova or parasites seen.11 show examples of some of these registers.5–2.

Secondly. and for the preparation of the budget for laboratory services at the national level. it helps to keep a check on the laboratory’s activities and is useful for ensuring adequate staffing.48 Manual of basic techniques for a health laboratory Firstly. An example of a monthly report is given in Table 2. for the ordering of supplies by the central stores. a monthly report is an aid in public health surveillance of the area covered by the laboratory since it reports the number of positive results obtained for various communicable diseases. Reports based on the number of tests done are the most suitable.12. .

1. substance concentration (mmol/l)”. E: eosinophils.1. In that case the column heading would read “reticulocyte no.0 2. concentration” and the values would depend on the erythrocyte number concentration (not reported in the examples given). respectively.1.35 0.6 mmol/l.01 2 Mr G Dr W 49 Table 2. of P. few hyaline cysts Leukocytes (5–10 per high-power field). ANS: anisocytosis. In that case. Setting up a peripheral health laboratory Hb: haemoglobin.3 Results sent (date) Date 12.Table 2.32 0. L: lymphocytes. M: monocytes.04 — Many P. O: other.1.01 2 Mrs E Dr A 6.6 Blood chemistry register Glucose concentration (mmol/l) Other tests (specify) — — 2. c The reticulocyte concentration may also be reported in terms of the number concentration.1. Morphology fraction (mm/h) concentration 2. In that case the example quoted (no.8 — Specimen no.01 0. For explanation of the column headings. b Haemoglobin may also be reported in terms of the substance concentration. the column heading would then be “Hb (Fe). +: weakly positive. . MEHC: mean erythrocyte haemoglobin mass concentration. POIK: poikilocytosis.01 1 Mr C Dr R 7.01 — 23 — ANS ++ POIK + PMN ++ 124 ¥ 10 — 4.1. Patient Sent by Urea. few epithelial cells +++ Test for protein Negative Test for bile pigments ND Test for urobilinogen ND Test for ketones ND Chemical test for blood ND Other tests (specify) ND Results sent (date) 2. i.56 0. see the relevant sections of the text. ++: moderately positive.08 — Moderate no.1.1. HC: hypochromic erythrocytes. concentration Type no.1.13 0.01 2. a Table 2.3 and 3. the values quoted in the two examples would be 7.2 ¥ 10 0.1.7 ¥ 109 -3 9 Date Specimen Patient Sent no.48 0.21 52 — 2 Mrs L Dr H 58 — 2. fractionc (g/l) Volume ESR No.e. Patient Sent by pH 2. ESR: erythrocyte sedimentation rate.1.01 1 Mr R Dr M 117 2. the column heading would then be “mean erythrocyte haemoglobin (Fe) concentration (MEH(Fe)C (mmol/l)”. +++: strongly positive. substance concentration (mmol/l) 2.01 ND: test not done. —: negative.01 Date Specimen no. by Hb concentrationb (g/l) Reticulocyte MEHCd no. falciparum trophozoites ANS ++ POIK ++ HC ++ PMN + 276 0. falciparum trophozoites 0.01 1 Mrs W Ward 1 2.01 2.1. N: neutrophils.5 Haematology registera Erythrocytes Leukocytes Malaria Other Results tests sent (date) No. the number per litre.01 — 5. d MEHC may also be reported in terms of the substance concentration. fraction N L M E O — 2.7 Urine analysis register Direct microscopic examination Test for glucose Negative Leukocytes (20–30 per high-power field). 2) would have a value of 17.8 Negative ND ND + ND Pregnancy test: positive 2.1. PMN: polymorphonuclear erythrocytes.071 ¥ 10-3 5.04 0.

01 2.01 Manual of basic techniques for a health laboratory 3.94. Patient Sent by Macroscopic appearance 2. fraction: neutrophils 0.1.3 0.1.5 30 Gram-staining shows many leukocytes and a few Gram-negative intracellular diplococci ND 4 3. of Gramnegative rods Moderate no.1.01 Date Specimen no.01 1 Mr J 2. including gonococcal cocci Occasional leukocytes and epithelial cells. of intracellular Gramnegative diplococci seen. concentration Glucose concentration (mmol/l) Total protein concentration (g/l) Pandy test for globulin + 0.1.50 Table 2.1.1.1.1. no erythrocytes or organisms seen Results sent (date) 2.1.45 1.1.01 Medical ward 1 CSF 4 Mrs R 3.01 Date Specimen no.01 3 Mr L 3. moderate no. few erythrocytes. lymphocytes 0.01 Table 2. few epithelial cells.1.8 CSF examination (in urine analysis register or separate) Direct microscopic examination Leukocyte no.01 1 Ms W Dr G Cloudy Leukocyte type no.9 Bacteriology register Sent by Specimen Sputum Pus from wound Urethral pus Dr R Medical ward 2 Dr M Examination requested Microscopic examination of smear for tuberculosis Microscopic examination of Gram-stained smear Microscopic examination of Gram-stained smear Microscopic examination of Gram-stained smear Results No acid-fast bacilli seen Many leukocytes.1.06 17. Patient 2.25 Negative Other tests (specify) Results sent (date) 2.01 .01 2 Mr L Dr C Clear ND 17.01 2 Mrs A 3.

1.1.1.01 3.1.1.01 ELISA: enzyme-linked immunosorbent assay.1.01 2 Ms M Dr C Stool 2.01 Specimen no.1.1. 1 Patient Mr F Sent by Dr A Specimen provided Stool Examination requested Intestinal parasites Intestinal parasites Results Direct microscopy: moderate no.1.01 Specimen no. 1 2 Patient Mrs P Mrs T Sent by Prenatal clinic Dr M Specimen Blood Blood Examination requested ELISA for determination of antibodies to HIV ELISA for determination of antibodies to HIV Results Non-reactive Reactive.10 Parasitology register Date 2.1. HIV: human immunodeficiency virus. Setting up a peripheral health laboratory 51 Table 2.1.11 Serology register Date 3.01 3.1.01 2.01 Results sent (date) 2.01 Table 2. 1 : 8 Results sent (date) 3.01 3.2. of Ascaris lumbricoides ova seen Direct microscopy: no ova or parasites seen Concentration technique: no ova or parasites seen 2.01 3. .01 3 4 Mrs L Mr S Medical ward 1 Dr R Skin snips Stool Onchocerciasis Parasites No parasites seen Occult blood: positive Direct microscopy: many trophozoites of Entamoeba histolytica and a few hookworm ova seen 3.

12 Sample monthly report for a health laboratory Name of laboratory: Report for the month ending: LABORATORY RECORD Number of examinations carried out Haematology (general) Blood chemistry Urine analyses: — direct examination — chemistry Pregnancy tests CSF examinations: — direct examination — chemistry Parasitology: — examination of stools — examination of blood — other examinations (e.52 Manual of basic techniques for a health laboratory Table 2.g. examination of lymph glands for trypanosomes) Bacteriology: — Gram stains — acid-fast stains — Wayson stains Mycology Serology: — qualitative — quantitative Number of specimens sent to specialized laboratories Water for bacteriological analysis Specimens for bacteriological culture Sera for serology Tissue biopsies Other specimens COMMUNICABLE DISEASES RECORDa Number of cases reported Gonorrhoea Leprosy Plague Tuberculosis Amoebiasis Ascariasis Filariasis Hookworm Malaria Onchocerciasis Schistosomiasis a 1235 27 287 43 17 3 3 162 802 2 63 41 11 3 114 16 8 32 0 2 0 11 0 0 7 14 22 1 80 253 0 2 The list of notifiable diseases varies from country to country. It is established by the central public health authority on the basis of: — international regulations on reporting communicable diseases — diseases prevalent in the area. .

General laboratory procedures 53 3. which gives a slow controlled movement to the object slide.1) This consists of: — the foot (1) — the limb (2) — the revolving nosepiece (objective changer) (3) — the stage (4) — the mechanical stage (5). Magnification system (Fig.1 Components of the support system of a microscope 1: foot. Fig.1. 5: mechanical stage. just above the preparation under examination (the object). and is called the objective. 3. 2: limb.1 Use of a microscope The microscope is an essential instrument for the diagnosis of disease. q The first group of lenses is at the bottom of the tube. 3. 3. It is a precision instrument and requires careful maintenance to prevent damage to the mechanical and ocular parts and also to stop fungi from obscuring the lenses. The second group of lenses is at the top of the tube and is called the eyepiece. The lenses of the microscope are mounted in two groups.1 Components of a microscope The various components of the microscope can be classified into four systems: — the support system — the magnification system — the illumination system — the adjustment system. q Objectives Magnification The magnifying power of each objective is shown by a figure engraved on the sleeve of the lens (Fig. General laboratory procedures 3. 4: stage. 53 .3): — the ¥ 10 objective magnifies 10 times. 3: revolving nosepiece. — the ¥ 40 objective magnifies 40 times. 3. Support system (Fig. — the ¥ 100 objective magnifies 100 times. 3.2) This consists of a system of lenses.3. one at each end of the long tube — the body tube.

) Some microscopes are fitted with a ¥ 3 or ¥ 5 objective instead of a ¥ 10 objective.2 Components of the magnification system of a microscope Fig. for example: Fig. 3. 3.4). next to the magnification (Fig.54 Manual of basic techniques for a health laboratory Eyepiece Tube Objective wh o 01 24 3 Fig.4 Numerical aperture . 3. Numerical aperture The numerical aperture is also engraved on the sleeve.3 Objective lenses (The ¥ 100 objective is usually marked with a red ring to show that it must be used with immersion oil. 3.

the smaller the working distance (Fig. The maximum resolving power of a good medical laboratory microscope is about 0. Moreover. The screw threads of all objectives are standard. The front lens of the ¥ 100 objective is the size of a pinhead.16 mm.25 mm (the resolving power of the normal human eye is about 0. 0. 3.30 on the ¥ 100 objective. the greater the numerical aperture.25 mm).5): — ¥ 10 objective: the working distance is 5–6 mm — ¥ 40 objective: the working distance is 0.5 Working distance of an objective Resolving power The resolving power of an objective is its ability to reveal closely adjacent details as separate and distinct.5–1. the clearer the image. the smaller the front lens mounted at the base of the objective. .g. The greater the numerical aperture.3. so handle it with care. Immersion oil increases the resolving power by conserving many light rays that would be lost by refraction if a dry objective were used. — the recommended thickness in millimetres of the coverslip used to cover the object slide — e.65 on the ¥ 40 objective — 1. 3. so the objectives in the revolving nosepiece are interchangeable. The greater the resolving power of the objective. Fig. The greater the magnifying power of the objective. Other figures that may be marked on the sleeve The sleeve may also display: — the recommended length in millimetres of the tube (between the objective and the eyepiece) — usually 160 mm.5 mm — ¥ 100 objective: the working distance is 0. Working distance The working distance of an objective is the distance between the front lens of the objective and the object slide when the image is in focus.15–0.20 mm.30 on the ¥ 10 objective — 0. the greater the resolving power (see below). General laboratory procedures 55 — 0.

multiply the magnifying power of the objective by that of the eyepiece.6 An eyepiece Fig. One side has a plane surface. But the contrast is correspondingly diminished. 3. the total magnification is: 5 ¥ 40 = 200. 3.6): — a ¥ 5 eyepiece magnifies the image produced by the objective five times. 3. It is situated between the mirror and the stage. since it is easy to adjust. 3. If the object is magnified 40 times by the ¥ 40 objective.56 Manual of basic techniques for a health laboratory Eyepiece Magnification The magnifying power of the eyepiece is marked on it (Fig. 3. 3. 3. To calculate the total magnification of the object observed. the other a concave surface (Fig. The concave side forms a low-power condenser and is not intended to be used if the microscope already has a condenser. Fig. essential for using the ¥ 100 objective. Fig. is used to reduce or increase the angle and therefore also the amount of light that passes into the condenser.9). Mirror The mirror reflects rays from the light source onto the object. Diaphragm The diaphragm (Fig. however. The condenser can be raised (maximum illumination) and lowered (minimum illumination). which is inside the condenser. Certain eyepieces have a calibrated graticule. protozoan cysts).8) brings the rays of light to a common focus on the object to be examined. or by an external lamp placed in front of the microscope. .7). Illumination system Light source An electric light source is preferable. It must be centred and adjusted correctly.7 A microscope mirror Condenser The condenser (Fig.g. then by five times by the ¥ 5 eyepiece. Electric illumination is. These eyepieces are used to measure the size of an object under the microscope (e.8 A condenser The wider the diaphragm the greater the numerical aperture and the smaller the detail seen. Binocular microscopes Binocular microscopes (two eyepieces but using only one objective at a time) are generally recommended. — a ¥ 10 eyepiece magnifies the image 10 times. It is provided either by a lamp built into the microscope beneath the stage. Microscopes used in medical laboratories have a magnifying power of between ¥ 50 and ¥ 1000. They are less tiring for the eyes than monocular microscopes when long examinations have to be made.

3: condenser adjustment screw. Adjustment system (Figs. It is used to bring the object into perfect focus. 3. 3. Coarse adjustment screw This is the largest screw.10 and 3.10 Microscope adjustment system 1: coarse adjustment screw. 4: condenser centring screws. 5: iris diaphragm lever.9 A diaphragm Filters In some microscopes coloured filters (particularly blue filters) are fitted below the condenser. 3. 2: fine adjustment screw. These can be left in place or removed according to the type of preparation being examined.3. Fig. Fig. Fine adjustment screw This moves the objective more slowly. General laboratory procedures 57 Fig.11) This consists of: — a coarse adjustment screw — a fine adjustment screw — a condenser adjustment screw — condenser centring screws — an iris diaphragm lever — mechanical stage controls.11 Mechanical stage controls . It is used first to achieve an approximate focus. 3.

They are used to centre the condenser exactly in relation to the objective. 3. A porcelain holder for a light bulb is fixed on a wooden base and the whole is encased in a wooden or tin box with an opening for the light (Fig. The microscope must be placed in the shade away from the window. a flap can be fitted above the opening to serve as a shutter (Fig. Iris diaphragm lever This is a small lever fixed to the condenser.2 Setting up the microscope When a new microscope is received in the laboratory.1. use a piece of heavy cloth. following this order in a clockwise direction: — ¥ 3. Mechanical stage controls These are used to move the object slide on the stage: one screw moves it backwards and forwards and the other screw moves it to the left or right (see Fig. Fitting the accessories Screw the objectives into the revolving nosepiece. Use a 100 W opaque electric bulb of the “daylight” type (blue–white). Alternatively. After you have screwed in the objectives: q q q Put the eyepiece(s) in place. Fix the mirror on the foot. Positioning the microscope Place it on a firm level bench (check with a spirit level) of adequate size but not too high. 3. Fix the condenser under the stage. it is important to know how to set it up correctly. you can make a lamp to provide illumination. Place a square felt pad under the microscope. 3.13).58 Manual of basic techniques for a health laboratory Condenser adjustment screw This is used to raise the condenser for greater illumination or to lower it to reduce the illumination. one on the left and one on the right. ¥ 5 or ¥ 10 objective. Condenser centring screws There may be three screws placed around the condenser: one in front. The screw threads are standard.11). . — ¥ 40 objective. It can be moved to close or open the diaphragm. If no felt is available. — ¥ 100 oil-immersion objective. thus reducing or increasing both the angle and the intensity of the light. 3. Cut slits in the top of the box to enable the bulb to cool. Setting up a lamp for the microscope If the microscope has a mirror.12).

Adjust the position of the lamp so that it shines on the centre of the mirror (Fig. 3.3. 3. 3. General laboratory procedures 59 Fig. 3.13 Alternative light source for the microscope Positioning the lamp If electric illumination is to be used. place the lamp 20 cm in front of the microscope facing the mirror.12 Setting up a lamp for the microscope Fig.14).14 Positioning the light source . Fig.

1. Raise the condenser slowly until the edges of the circle of light are in sharp focus (Fig. Raise the condenser. first close the diaphragm It is very important to centre the condenser correctly. 3.16). 3. This is often overlooked. Place a slide preparation without a coverslip on the stage. Adjust the position of the mirror (if necessary) so that the circle of light is in the exact centre of. Place a piece of thin white paper over the lens at the top of the condenser (Fig. The piece of paper should show an image of the electric bulb. as seen through the condenser Centring the condenser (if centring is provided for) Fig. 3. surrounded by a circle of light.17 To centre the condenser. ¥ 5 or ¥ 10).19). Close the diaphragm.15 Adjusting the mirror Fig.17). Fig. 3.18). 3. 3. or superimposed upon. the filaments of the bulb are projected on to a piece of paper covering the mirror. Remove any coloured filters. Open the iris diaphragm.60 Manual of basic techniques for a health laboratory If the lamp is fitted with a lens. Look through the eyepiece and bring the slide into focus. A blurred circle of light surrounded by a dark ring appears in the field (Fig. Examine with the lowest-power objective (¥ 3. Then check with the other objectives. Preliminary adjustment of the mirror Use the plane side of the mirror.16 Image of the light source. 3.20). Adjust the centring screws of the condenser so that the circle of light is in the exact centre of the field (Fig. This makes it possible to centre the beam more precisely. 2.18 Raise the condenser until the edges of the circle of light are in focus . Fig. adjust the mirror so as to maximize the amount of light passing through the condenser. If daylight is being used. Lower the condenser. 3. 4. Open the iris diaphragm to the maximum. 3.15). 3. 3. Adjust the mirror so that the image of the bulb is in the exact centre of the circle of light (Fig. the bright area surrounded by the dark zone (Fig. In some models the bulb is turned until a clear image of the filament is obtained. 5.

3. Fig. 3.21). Binocular adjustment When a binocular microscope is used.22).3 Focusing the objective Low-power objective (¥ 10) ¥ Rack the condenser down to the bottom. If the image is not clear. Close the diaphragm slowly until the circle of light takes up only two-thirds of the surface (Fig. Then close your right eye and look through the left eyepiece. If the image is in focus. bring the image into focus for your right eye with the right eyepiece. using the coarse adjustment screw. The eyepiece to use is a matter of individual choice. If the collar is on the left eyepiece holder. Fig. Do this for each objective as it is used. the interpupillary distance (the distance between the pupils of the eyes) can be adjusted to suit the operator. 3. Raise the objective.21 Adjusting the diaphragm Fig. no adjustment is needed. Remove the eyepiece and look down the tube: the upper lens of the objective will be seen to be filled with a circle of light. Focusing the eyepieces One of the eyepiece holders (usually the left) has a focusing collar (Fig.3. 3. Adjusting the eyepieces Selecting the eyepiece The ¥ 5 and ¥ 10 eyepieces give good results in the medical laboratory.19 Adjust the position of the mirror to centre the light source Fig. using the ¥ 40 objective. The microscope is now adjusted to suit your own binocular vision. close your left eye and. General laboratory procedures 61 Adjusting the diaphragm Open the diaphragm completely. 3. turn the focusing collar until it is in focus. Lower the objective until it is just above the slide preparation. until a clear image is seen in the eyepiece. 3.22 Focusing the eyepieces .1.20 Use the centring screws of the condenser to centre the light source 3. The highpower eyepiece increases magnification but there may be no great increase in detail.

3.g. How to establish the position of images seen Images observed in the microscopic field can be placed in relation to the hands of a clock. Objects seen on the left side of the microscopic field are actually on the right. If the illumination is inadequate. Rack the condenser up slightly if there is insufficient illumination. Lower the ¥ 100 objective until it is in contact with the oil. Lower the objective until it is just above the slide preparation (the working distance is very short — about 0. in preference to cedarwood oil. Raise the condenser to obtain sufficient illumination. Depth of the microscope field The image is seen in depth when a low-power objective is used. use the concave side of the mirror as recommended for the ¥ 40 objective. Using the coarse adjustment screw. which dries quickly). 3. and open the iris diaphragm fully. Bring it as close as possible to the slide. a schistosome egg is placed at “2 o’clock” in Fig. Inversion of images The image seen is inverted by the lenses: q q Objects seen at the bottom of the microscopic field are actually at the top. If the microscope has no condenser. 3.23. Oil-immersion objective (¥ 100) ¥ Perfectly dry. it is not the objective holder but the stage which is moved up and down by the coarse and fine adjustment screws to bring the image into focus. Fig. Rack the condenser up as far as it will go. raise the objective very slowly until a blurred image appears in the field. use the concave side of the mirror. the different nuclei in a spherical amoeba cyst).23 Establishing the position of images seen under the microscope Moving the object If you move the slide in one direction. . This is because the fine adjustment screw has been turned right to the end. but avoid pressing on the preparation (modern objectives are fitted with a damper).24). Images seen under the microscope Remember that the circle of light seen in the eyepiece is called “the microscopic field”. High-power objective (¥ 40) ¥ Rack the condenser half-way down. the depth of focus is small and the fine adjustment screw must be used to examine every detail from the top to the bottom levels of focus of the object observed (e.5 mm). Turn it back as far as it will go in the other direction and then focus by raising the objective.62 Manual of basic techniques for a health laboratory Occasionally a clear image cannot be obtained although the objective has been lowered as far as possible. ¥ 100) are used. Bring into focus using the fine adjustment screw. For example. Important: In most modern microscopes. stained preparations must be used. Place a tiny drop of immersion oil on the part to be examined (use synthetic oils. which do not dry. the object examined moves in the opposite direction (Fig. When the highpower objectives (¥ 40. Look through the eyepiece and turn the fine adjustment screw very slowly upwards until the image is in focus.

2.26 Calibration of an ocular micrometer with a stage micrometer . 3. Look for another set of lines where the scale of the stage micrometer coincides with that of the ocular micrometer.3.26). Use lens paper to handle the disc. 3. Put the calibrated stage micrometer on the stage of the microscope and focus on the scale. 5. 3.1-mm and 0. Count the number of 0.4 Use of an ocular micrometer The size of microorganisms or substructures of organisms can be measured by microscopy using an ocular with a calibrated micrometer disc. Count the number of subdivisions of the ocular micrometer scale between the 0-line and the second set of coinciding lines. This set of lines should be as far away from the 0-mm line as possible (Fig. 3. 10. so that it is not lost after changing the objective. depending on the magnification of the objective of the microscope. Replace the lens carefully.01mm subdivisions. Materials q q q q q q Fig. 3. Adjust the stage micrometer so that the 0-mm line coincides with the 0-mm line of the ocular micrometer. Unscrew the eye lens of the ocular. 3.25 An ocular micrometer disc Fig. Method 1. raise the nosepiece before changing to the more powerful objective and refocus. If this is not the case for your microscope. make sure that the object examined is in the middle of the field. 6. 8.1. 4. You should be able to clearly distinguish the 0. Place the micrometer with the engraved scale face-down in the ocular. Before changing objectives. 9. 3.24 Moving the object Binocular microscope Ocular with a ¥ 10 magnification Ocular micrometer disc Stage micrometer Lens paper Immersion oil.1-mm subdivisions of the stage micrometer scale between the 0-line and the second set of coinciding lines. The distance between the two coinciding sets of lines varies. The micrometer disc has a scale that is usually divided into 0. Calculate the proportion of a millimetre that is measured by a single ocular unit using the following formula: stage reading (mm ) ¥ 1000 mm = ocular units (mm ) ocular reading ¥ 1mm Fig.25). A stage micrometer is used to calibrate the ocular micrometer. Place the ocular with the micrometer in the ocular tube of the microscope.01-mm subdivisions (Fig.1-mm and 0. General laboratory procedures 63 Changing the objective Modern microscopes are made so that the object remains more or less in focus when you change from a low-power objective to a more powerful one. 7.

a soft camel-hair brush (or a fine paintbrush or blower for cleaning lenses) A desiccator 15–20 cm in diameter containing not less than 250 g of dry blue silica gel (which indicates humidity by turning pink).6 Routine maintenance Microscopes must be installed in a clean environment. if possible. The ocular containing the micrometer disc should be stored until required. Each microscope that is to be used for measuring the size of organisms must be individually calibrated.1.1. . The microscope needs daily attention to keep it in good working order and thus to ensure reliable laboratory results. The stops must be made of a material through which light cannot pass and must be the correct size for the objective in use. Cleaning the microscope Microscopes are used to investigate biological tissues and fluids and must therefore be decontaminated at regular intervals. 3. white absorbent paper or medicalgrade cotton wool A piece of chamois leather. Workplaces should be well ventilated or permanently air-conditioned (intermittent use of air conditioners produces condensed water). the calculation is as follows: 0. q q q q q Method Cleaning the optical surfaces The optical surfaces (condenser.64 Manual of basic techniques for a health laboratory Example For a microscope with a high-power objective (¥ 40). Optical instruments should not be kept for long periods in closed compartments since these conditions also favour fungal growth which can corrode optical surfaces. If the stop is too small. a soft camel-hair brush (Fig.5 Dark-field microscopy To obtain a dark field a special condenser with a blacked-out centre is used. too much light will pass into the objective and a dark field will not be obtained. Special care is required in hot and humid climates. If this is not available it is possible to obtain a dark field under the ¥ 10 and ¥ 40 objectives by inserting a disc or stop in the filter holder below the condenser. Materials q q Clean pieces of old cloth and a fine linen handkerchief Special lens tissue paper or. 3. unscrew the upper lens and clean the inside. If dust is found inside the eyepiece. if possible (otherwise a non-fluffy rag) A small bottle of cleaning solution (see below) A plastic cover A small rubber bulb and.27) or a blower. eyepieces) must be kept free of dust with a fine paintbrush. insufficient light will be available to illuminate the specimen. away from chemicals. objectives. 3. if unavailable. If the stop is too large.1mm ¥ 1000 mm = 2 mm 50 units ¥ 1mm Important: Corresponding objectives should not be exchanged for a calibrated objective. but must be separately calibrated.

This mixture is not suitable for cleaning the optical surfaces. Check the spring load on the specimen clamp. Cleaning the instrument Heavy contamination can be removed with mild soapy solutions. Too high a tension may result in breakage of slides and damage to the clamp. Never press the objective on to the slide. Check the focusing mechanism. take care not to confuse the condenser centring screws with the condenser clamp screws. Never leave the microscope without the eyepieces unless the openings are plugged. Grease and oil can be removed with the special cleaning solution described above. since both the slide and the objective may break. be used for cleaning mirrors. however. Clean all mechanical parts. Note: Do not use 95% ethanol. Lubricate the microscope according to the manufacturer’s instructions. Never use ordinary paper to clean the lenses. Check the optical alignment. absorbent paper or medical-grade cotton wool. The instrument should then be cleaned with a 50 : 50 mixture of distilled water and 95% ethanol. Check the diaphragm. fine adjustment screw. a fine paintbrush or a blower instead. Never keep the microscope in a closed wooden box in hot humid countries. condenser focusing and mechanical stage) should be periodically cleaned and lubricated with machine oil to make them run freely. A dim appearance of the specimen is often due to misalignment of the optical parts rather than to insufficient light. q Precautions q Never dip the objectives in xylene or ethanol. Never clean the inside lenses of the eyepieces and objectives with cloth or paper (this might remove the anti-reflective coating). To maintain the microscope proceed as follows: q q q q q q q Fig. xylene or toluene for cleaning the lenses. They can. General laboratory procedures 65 Oil residues on the lenses should be removed with special lens tissue paper. q q q q q q q . Never touch the lenses with your fingers. use a soft camel-hair brush. Remove any fungal growth. The optical surfaces may be finally cleaned with a special solution. Maintaining the microscope When you carry out repair and maintenance procedures. consisting of the following: — 80% petroleum ether (boiling point 60–80°C) — 20% 2-propanol.27 Cleaning the objective lenses using a soft camel-hair brush Check the mechanical stage. as this may cause the lenses to become detached.3. Take care when focusing the microscope. since these substances dissolve the cement. The mechanical parts (coarse adjustment screw. Never clean the support or the stage with xylene or acetone. 3.

The balance is used to weigh chemicals for production of reagents. This can be prevented as described below. a fine paintbrush or a blower. particularly on the surface of the lenses. Remove stains or chemicals using a soft brush. as this may cause misalignment. adjust the voltage with a dimmer switch to give the lowest required light intensity. The optical surfaces should be cleaned with lens cleaning tissue or soft tissue paper. If the mains voltage fluctuates excessively. Finish cleaning the lenses with a soft camel-hair brush. This can be avoided as follows: q q Always keep the microscope under an airtight plastic cover when not in use. Never carry the microscope by the limb with one hand. in the grooves of the screws and under the paint. Never put the microscope away with immersion oil on the objective. Mild soap solution is suitable for most cleaning. q Humid climates In hot. . These procedures must be carried out regularly. as fingerprints reduce the intensity of illumination. clean it with special lens tissue paper. dry climates. use a voltage stabilizer. and are essential in conjunction with repair and maintenance procedures. never place chemicals directly on to the pan. clean the microscope thoroughly by blowing air over it with a rubber bulb. Always keep the microscope under an airtight plastic cover when not in use.66 Manual of basic techniques for a health laboratory q q Keep the mechanical stage clean. Use organic solvents only in accordance with the manufacturer’s recommendations. Use a plastic weigh boat or filter-paper to weigh chemicals on the balance. 3.2 Weighing: use of laboratory balances Balances may be either electrically or manually operated. To maximize the lifespan of bulbs. dry climates the main problem is dust. All types should be positioned on a firm level bench away from vibrations. Do not dismantle the optical components. q q q q q q Additional precautions to be taken in hot climates Dry climates In hot. If dust particles remain on the surface of the objective. fungi may grow on the microscope. Remove any oil daily. and cleanliness is essential if accurate results are to be obtained: q q q Remove dust by blowing or using a soft brush. draughts and direct sunlight. It can be simply regenerated by heating in a hot-air oven or over a fire. one under the foot. humid climates and during the wet season in hot. At the end of the day’s work. (The silica will turn red when it has lost its capacity to absorb moisture from the air. together with a dish filled with blue silica to dry the air under the cover. use both hands. avoid touching the glass with your fingers. Fine particles work their way into the threads of the screws and under the lenses.) The microscope must be cleaned daily to get rid of dust. the other holding the limb. and the instrument will soon be useless. When changing a bulb.

5 g. Instructions for use 1. It may be designed for use with separate weights. the two circles should be of equal weight. 22.2 Open two-pan balance (Fig. Handle loose weights with forceps.5 g.1 Sensitivity of a balance The sensitivity corresponds to the smallest mass that makes the pointer move over one division on the scale. 38 g.3. (Use a clean spatula to dispense small amounts of substances for weighing. 3. 3.29. To measure out the substance to be weighed. Place the bottle containing the substance to be weighed to the left of the balance. Place on the right-hand pan the weights equivalent to the weight of the receptacle plus the amount of the substance to be weighed. General laboratory procedures 67 Important: If water has been used to clean the balance.380 g. If the pans are made of easily scratched or corroded material. 4. For example.30). 8. Place on the left-hand pan the receptacle (folded paper or dish) in which the substance will be weighed. Fig.5 g. Always set the balance to zero before weighing. if the sensitivity of a balance is 1 mg. e. so that the powder or crystals to be weighed fall little by little into the receptacle (Fig. Check the accuracy of the balance regularly according to the manufacturer’s recommendations. For routine laboratory purposes. the sensitivity of a balance can be considered to be the smallest mass that it will measure accurately.28 An open two-pan balance Fig.29 Set of weights (in grams) for use with an open two-pan balance . hold the bottle tilted in your left hand (label upwards) and tap the neck of the bottle gently with your right hand.2. 3.2. this means that a mass of at least 1 mg is needed to move the pointer.31).28) The two-pan balance has two pans supported by shafts. protect them with circles cut out of strong plastic or old X-ray films.) When the substance has been weighed. move the bottle to the right-hand side of the balance (Fig. It is used to weigh large amounts (up to several kilograms) when a high degree of accuracy is not required. as illustrated in Fig. 3. 3. Sensitivity: 0. 3. or may incorporate a graduated arm with a sliding weight. make sure that it is thoroughly dry before weighing. 3. 2.g. 3. 3.

Beam release screw (or pan lock control) (B). This is the structure from which the pans are suspended. . These support the beam at the fulcrum during the weighing and give sensitivity to the balance. — after weighing. 6. q q q q q Figure 3. S2). 3. KE3).68 Manual of basic techniques for a health laboratory Fig. 0. Pointer (Pt).32) q q Cross-beam (CB). Stirrups (S1. 3. Used only for initial adjustment of the unloaded balance to a reading of zero.1–0. KE2. AS2).85 g.2.3 Analytical balance This balance has two pans suspended from a cross-beam inside a glass case.220 g. Pans (P). when you move the bottle to the right of the balance. 3. depending on the model). Use the balance: — to weigh small quantities (up to 20 or 200 g.5 mg. Sensitivity: 0.30 Measuring out the substance to be weighed Fig. Read the label three times: — before taking the bottle off the shelf. — when great accuracy is required: e. Locks the pan so that the sudden addition of weights or chemicals will not damage the delicate knife edges. Knife edges (KE1. 3. This avoids confusion. Instructions for use q Always ensure that the cross-beam is at rest (beam release screw tightened) before placing the weights and the substance to be weighed on the pans. Components (Fig. Adjusting screws (AS1. Those on the beam support the suspended pans.33 shows a set of weights for use with an analytical balance.740 g.g. — while weighing the substances (label facing upwards). 3.31 Keep weighed and unweighed substances apart to avoid confusion Thus place: — the weighed substances on the right — the unweighed substances on the left. depending on the model.

if the radius is 25 cm and the rpm is 1300 rev/min. KE2. General laboratory procedures 69 Fig. 50 mg. Always put the cross-beam back at rest before removing the weights and the substance that has been weighed from the pans. 3.3. 10 g. 3. S2: stirrups.35). Fig.35 Principle of centrifugation . Fig. 3. CB: cross-beam. 3. q Check that the pans are balanced (after closing the glass case) by unscrewing the beam release screw. KE3: knife edges.4 Dispensary balance (Fig. Use the adjusting screws (AS1 and AS2) to obtain a perfect balance when compensating for the weight of the receptacle in which the substance will be weighed.2. q q q q 3.1 Principle A body is rotated in a circular movement at speed. 5 mg. 200 mg and 500 mg. To calculate the relative centrifugal force (rcf) for an individual centrifuge. or in a watch glass or porcelain dish. The dispensary balance is more accurate than the open two-pan balance. 5 g. S1. Always use forceps to pick up the weights. AS2: adjusting screws. the rcf is about 50g. 3. put it away in a closed cupboard. 10 mg.33 Set of weights for use with an analytical balance Single pieces: 1 g. Always place the substance to be weighed on a piece of paper folded in four. Single fractional pieces: 2 mg. 50 g.34) This balance also has two suspended pans. but weighs only up to 50 g. measure the radius (r) of the rotor arm (in cm) and the number of revolutions per minute (rpm) and use the formula below: rcf = 1. P: pans. 2 g.118 ¥ 10-6 ¥ r ¥ (rpm)2 For example. Sensitivity: 5–10 mg. 20 g.3. 100 g. This creates a force that drives the body away from the centre of the circular movement (Fig.32 Components of an analytical balance AS1. 100 mg.34 A dispensary balance 3. 20 mg.3 Centrifugation 3. but it has no glass case and no supports. B: beam release screw. KE1. 3. Fig. After using the dispensary balance. 200 g and 500 g. Pt: pointer.

Lubricate the spindle of the centrifuge regularly. The speed is insufficient for satisfactory separation of erythrocytes from plasma in blood. The particles are compacted at the bottom of the centrifuge tubes. section 3. — parasite eggs (in diluted stools). so follow the instructions above carefully.38) The head is designed to swing the tubes out to a horizontal position during centrifuging. Pull the handle out of the machine with a sharp movement. It is useful for certain techniques. To stop the centrifuge. e. 3.g. — to concentrate certain parasites in stools. T: centrifuge tubes. fixed to the shaft.2 Types of centrifuge Hand-operated centrifuge (Fig. Electric centrifuges Electric centrifuges are more accurate than hand-operated centrifuges and should be used whenever possible. 3. 3.37 A hand-operated centrifuge q q q q Warning: The hand-operated centrifuge can cause serious injury.36) A centrifuge consists of: — a central shaft or spindle (A) that rotates at high speed. They swing out to the horizontal and the particles in suspension in the liquids in the tubes are thrown to the bottom of the tubes. When the spindle rotates the tubes are subjected to centrifugal force. Electric centrifuges are used with two types of head — the “swing-out” head and the “angle” head. Fig.37) This is operated manually by turning a handle. E: centrifuge head. These particles form the centrifuge deposit which can be separated from the supernatant fluid and examined. .3. Balance the two diametrically opposite tubes perfectly as described in the instructions for use.36 Components of a centrifuge A: central shaft or spindle. The deposit may contain.3. “Swing-out” head (Fig. Important: q q Clamp the centrifuge firmly on a stable support (edge of a table). 3. — tubes (T) containing the liquid to be centrifuged. Remove the tubes slowly and carefully (so as not to disturb the deposit). — a head (E). “Angle” head (Fig.39) The “angle” head holds the tubes at an angle of about 45° during centrifuging. 3. with buckets for holding the centrifuge tubes. for example: — blood cells. — cells from the urinary tract (in urine). Fig. Keep your distance while operating the centrifuge. This is the type most frequently needed.70 Manual of basic techniques for a health laboratory Components of a centrifuge (Fig.3. The hand-operated centrifuge can be used: — to examine urinary deposits. agglutination tests in blood-grouping by the testtube method. 3. It takes two or four tubes. do not slow down the turning of the handle. 3.

etc.3. 3.38 A centrifuge with a swing-out head Fig. 3.40 Types of bucket for a centrifuge Buckets (tube holders) There are several types of bucket for use with electric centrifuges (Fig.3.e. — buckets that hold nine small (precipitin) tubes. after 5 or 10 minutes).40). a dial with a needle that indicates the speed of the machine during centrifuging (this is useful for some methods of concentration of parasites). General laboratory procedures 71 Fig. 3.39 A centrifuge with an angle head Fig. The choice depends on the model of centrifuge: — buckets designed to hold one round-bottomed or conical tube only. 3. — a cooling chamber that prevents heating of the specimen during centrifuging. i. Some models are also fitted with: — a timer that stops the centrifuge automatically when the time is up (e. — buckets that hold two round-bottomed or conical tubes. Battery-operated centrifuges Small battery-operated centrifuges are sometimes used to measure the packed cell volume in haematology.3 Instructions for use You should always follow the manufacturer’s instructions when using the centrifuge. . 3. — a revolution counter.g.

until the desired speed is reached. Fig. When starting the centrifuge. If only one tube of liquid is to be centrifuged. 3. Stop the centrifuge gradually (some models have a brake that can be applied). Fill the tubes to no more than three-quarters full to prevent spillage in the bowl. turning the knob slowly. Remove the tubes slowly and carefully.42). 3. Fig. add a little water between each tube and its bucket. Never try to slow the centrifuge down with your hand. or add water to the bucket containing the lighter tube (using a wash bottle. This protects the bottom of the centrifuge tubes. Fig. 3. Ensure that the lid is closed before starting the centrifuge. gradually increase the speed. Failure to do this can cause excessive wear or the centrifuge may move. Never open the centrifuge until it has come to a complete stop. Tubes that are too long or too small may break.43 Balancing centrifuge tubes by adding water to the bucket containing the lighter tube To balance: either add more of the liquid to be centrifuged to the lighter tube (Fig.72 Manual of basic techniques for a health laboratory Installing the centrifuge The centrifuge must be placed on rubber pads or a mat on a flat level surface.43). Balancing the tubes If the tubes are numbered. Preventing breakage of tubes Always pad the bottom of the buckets with the rubber cushions provided with the machine.41: — tube 1 opposite tube 2. 3. 3. 3. Using a wash bottle. balance it with an identical tube filled with water. Safety precautions q Check that the tubes are the correct size for the centrifuge.41 Balancing centrifuge tubes Balance the tubes that are opposite each other by weighing them in their buckets on the open two-pan balance.42 Balancing centrifuge tubes by adding liquid to the lighter tube Fig. — tube 3 opposite tube 4. q q q q q q q . place them as shown in Fig. Always balance the centrifuge buckets before starting the centrifuge.

After discharge of the contents. according to its size (marked on the bulb) and the last drop is expressed against the side of the recipient container. q 3. It should not be expelled.0 ml can be dispensed. Many of the new procedures for analysis require very small volumes of fluid and various pipetting and dispensing devices are available to enable small volumes to be measured with great precision. q Various volumes can be measured using graduated pipettes. corrosive or poisonous. A volumetric flask measures a single volume of fluid. the pipette is allowed to drain for 15– 45 seconds. There are two types of graduated pipette (Fig.g. B: pipette with graduations not extending to the tip.44 A graduated pipette A pipette with graduations to the tip (A). Small volumes of fluid (0. For example: — a 10-ml pipette can be used to measure 8.5. There are two types of volumetric pipette (Fig. which is intended to be filled to the mark.45): q Fig.1–10 ml) can be dispensed rapidly and accurately using one of the following methods: q A fixed or variable volume dispenser attached to a reservoir made of glass or polypropylene. A measuring cylinder measures various volumes of fluid but is not very accurate.6 ml.46): q A pipette with a single graduation mark (A). 3.45 Types of graduated pipette A: pipette with graduations to the tip. — a 5-ml pipette can be used to measure 3. — a 1-ml pipette can be used to measure 0. It is important for the prevention of accidents that the correct procedures for the measurement and dispensing of these liquids are clearly understood and are followed conscientiously. 3.0 ml and from 2. 1 litre. 3. A pipette with graduations not extending to the tip (B). A calibrated pipette with a rubber safety bulb. The total volume is contained between the 0 mark and the last mark before the tip (this type is recommended for quantitative chemical tests). . see section 3. 3. Large volumes can be measured using a measuring cylinder or a volumetric flask.2 ml. General laboratory procedures 73 Cleaning and maintenance For details of cleaning and maintenance of centrifuges. e.44): — the total volume that can be measured. 3. Fig. — the volume between two consecutive graduation marks.4 Measurement and dispensing of liquids Many of the liquids handled in the laboratory are either infectious. Various volumes from 0.1 to 1.3.3. The total volume that can be measured is contained between the 0 mark and the tip.5 ml.0 to 10.4.1 Pipettes Types of pipette Graduated pipettes Graduated pipettes have the following information marked at the top (Fig. 3. Volumetric pipettes Volumetric pipettes are intended to measure a precise volume with a high degree of accuracy. accurately.

3. Micropipettes Micropipettes with disposable tips are frequently used to measure small volumes. 3. S: safety bulb. They are available in a variety of volumes.46 Types of volumetric pipette A: pipette with a single graduation mark. ranging from 5 ml to 1000 ml. Used tips are disposed of directly into disinfectant using an ejector mechanism. 3.48 A micropipette with a disposable tip T: disposable tip. Fig. They can be reused after disinfection and washing but cannot be autoclaved.48).74 Manual of basic techniques for a health laboratory q A pipette with two graduation marks (B) may be more accurate in skilled hands. 3. The tip of the pipette should be held against the side of the receptacle while the fluid is discharged. The micropipettes have two positions of the plunger operated by thumb (Fig. Plastic bulb pipettes Plastic bulb pipettes are cheap and very useful for transferring volumes of liquid such as serum or disinfectant. It is less reliable when used by an inexperienced person because it is easy to overrun the lower graduation mark when discharging the contents. Fig.The first position is used to pick up the sample and the second to expel the sample from the tip into a tube or well. Fig. . Hold the pipette vertically to check that the liquid reaches the desired graduation mark (G in Fig.47 How to hold a pipette G: graduation mark. 3. B: pipette with two graduation marks. This mark should be level with the bottom of the meniscus formed by the liquid. They are available with different tips and can be obtained with calibrations marked on the stem.47).

Hold the dropping pipette absolutely vertical to expel the drops (Fig.50) with the pipette instead. Precautions Pipetting by mouth is dangerous and should not be done. Calibration of dropping pipettes (Fig. 3.2 Volumetric flasks Volumetric flasks are graduated to measure a certain volume when filled to the graduation mark. Always use a rubber safety bulb (see Fig. Count the number of drops delivered from the millilitre of water. Draw the water into the dropping pipette to be calibrated.49). Micropipettes must be calibrated and maintained according to the instructions of the manufacturer.3. Repeat the procedure three times to check the accuracy. measure 1 ml of water into a small tube.4. . 3. thus 1 drop = 0.05 ml. Calibrated dropping pipettes Ordinary calibrated dropping pipettes often deliver 20 drops per ml of distilled water. 3. It can cause the following: — infection — burns — poisoning — cuts. 3. General laboratory procedures 75 Fig.49 Using a dropping pipette Fig. 3.50) Using a volumetric pipette (see page 74).50 Calibrating a dropping pipette S: safety bulb. 3.

Liquids expand with heat and contract with cold. into a 1000-ml flask through a funnel and diluting to the 1000-ml mark (Fig. 0. They should be used for the preparation of reagents. is prepared by washing 8. or cold liquids just taken from the refrigerator.51). 3. Stoppers Volumetric flasks should have plastic stoppers. Volumetric flasks are more accurate than measuring cylinders. Never measure hot liquids.52 Alternative method for preparing reagents using a volumetric flask For example: 1 litre of sodium chloride. 3.53 Mark the temperature at which the reagent should be measured on the flask . 3. 3. 3. Cost Volumetric flasks are very expensive. Fig. the substance(s) can be dissolved in a small container and the solution poured into the flask along a glass rod (Fig.76 Manual of basic techniques for a health laboratory They have various capacities: — 2000 ml — 1000 ml — 500 ml — 250 ml — 200 ml — 100 ml — 50 ml — 25 ml. Fill to the graduation mark.) Temperature of the liquid The temperature at which liquids should be measured is etched on the flask (after the capacity figure.53).51 Preparing sodium chloride solution in a volumetric flask Fig.5 g of sodium chloride. 3. Fig. Alternatively. so use them with great care. if these are not available use ground glass ones. dissolved in 100 ml of distilled water in a beaker. The solution should be shaken before use.52). 53). (This method is recommended for the preparation of titrated chemical reagents.85% solution (reagent no. Fig. Be careful not to lose them.

3. Rinse twice with ordinary water and once with demineralized water.5 Cleaning. 3. 25 ml and 50 ml capacity.55). Keep the top plugged or covered (Fig. 3.1 Cleaning glassware and reusable syringes and needles Instructions for cleaning: — glass containers (Erlenmeyer flasks. Burettes are filled from the top with the liquid to be measured (Fig. Dry. q q q .5.54). beakers. Avoid using them for laboratory tests.3 Burettes These are graduated glass tubes with a glass stopcock at the lower end. Fig.56) These are not accurate. In order to neutralize it: q Prepare a bowl containing 3 litres of water and 60 ml of concentrated hydrochloric acid (i. They are of 10 ml. Then insert the stopcock in the burette and rotate it until a smooth covering of the whole stopcock is obtained. Glass containers New glassware Glassware that has never been used may be slightly alkaline. To grease a clean stopcock properly.4.e. a 2% solution of acid). Maintenance of burettes The stopcock and tap should be kept well greased.54 Filling a burette 3. Fig.4 Graduated conical glasses (Fig.55 Keep the top of the burette plugged or covered Fig. apply the tiniest smear of petroleum or silicone jelly with a finger tip down the two sides of the stopcock away from the capillary bore. Leave the new glassware completely immersed in this solution for 24 hours. 20 ml.3.4. General laboratory procedures 77 3. disinfection and sterilization 3. 3. test-tubes) — pipettes — microscope slides — coverslips — reusable syringes and needles.56 A graduated conical testing glass 3. 3. 3.

3. preferably. Rinse each one thoroughly under the tap. Plugging The clean dry glassware should be put away in a cupboard to protect it from dust. Rinse each article in a stream of clean water. 3. reagents. Pipettes Immediate rinsing Once a pipette has been used. Drying Place the glassware in wire baskets and dry in a hot-air oven at 60 °C.58 Plug or cover glassware to protect it from dust .78 Manual of basic techniques for a health laboratory Dirty glassware Preliminary rinsing Rinse twice in cold or lukewarm water (never rinse bloodstained tubes in hot water). urine. serum. 3. Rinsing Remove the articles one by one. it should be rinsed immediately and then washed (never allow it to dry before rinsing). place the baskets in a sunny spot in the laboratory and cover them with a fine cloth. Soaking in detergent solution Prepare a bowl of water mixed with washing powder or liquid detergent. Place test-tubes upside-down in a wire basket.58) or. Put the rinsed glassware in the bowl and brush the inside of the containers with a test-tube brush (Fig. It is recommended that glass containers be plugged with non-absorbent cotton wool or their mouths covered with small caps made from newspaper (Fig.57 Cleaning dirty glassware Fig.) Draining Place containers (beakers. (Do not forget that traces of detergent left on glassware can lead to false laboratory results. Alternatively. etc. 3. Fig. rinse it immediately in a stream of cold water to remove blood. flasks. if available. If the glassware has been used for fluids containing protein.57). measuring cylinders) on the pegs of a draining rack. then soak them all in a bowl of ordinary water for 30 minutes. Leave to soak for 2–3 hours. thin sheets of paraffin wax or clinging plastic.

3. Microscope slides New slides Soaking in detergent solution Prepare a bowl of water mixed with washing powder or liquid detergent. If the pipettes have been used to measure infected material. Fig. Remove the pipettes one at a time. plastic measuring cylinder (or bowl) full of water. 20). Dip the other end of the pipette into the rinsing liquid (water or detergent solution). 3. 1. Soaking in detergent and rinsing Follow the instructions given above for soaking and rinsing of laboratory glassware. Rinsing in water Rinse each slide with tap water and then soak in clean water for 15 minutes. leave pipettes to air-dry. Alternatively. General laboratory procedures 79 Soaking in water After rinsing.59 Using a vacuum pump to rinse a pipette . 4. Rinse again.3. Fit this rubber tubing over the tip of the pipette. Put blocked pipettes in a cylinder filled with dichromate cleaning solution (reagent no. Using the vacuum pump This is a small instrument made of metal. Turn the water on hard to drive a strong jet through the pump. Check that the obstruction has been washed away. 3. place the pipettes in a large. leave them in a cylinder full of disinfectant solution (e. then change the water and soak for a further 30 minutes. Blocked pipettes 1. which is sucked through the pipette and discharged by the pump into the sink (Fig. 3.g. Slide them carefully into the solution and leave for 24 hours. 2. 5. plastic or glass that is attached to the water tap.59). wash at once with large quantities of water. pour the dichromate solution into another cylinder (it can be used four times). 2. Drying Dry heat-resistant glass pipettes in a hot-air oven at 60 °C and ordinary pipettes in an incubator at 37 °C. Use the amounts recommended by the manufacturer. Leave to soak in ordinary water for 30 minutes. a quaternary ammonium compound or 1% bleach solution. Warning: Dichromate cleaning solution is highly corrosive and should be used with extreme care. Place the slides in the bowl one by one and leave to soak overnight. This causes air to be sucked into the side arm of the pump and the rubber tubing attached to it. If it is accidentally splashed on the skin or clothing or into the eye(s). see pages 84 and 85) for 4 hours. The next day. Hold the cylinder containing the pipettes under the tap and rinse thoroughly.

Place them on a sheet of filter paper. Use the amount recommended by the manufacturer to produce a strong detergent solution. Remove the slides one by one from the strong detergent solution. pick the slides up by their edges. If you must use your fingers. for packets containing 20 slides each. 41–60. see overleaf). prepare another bowl containing a weak detergent solution (15 ml of household detergent per litre of water). non-fluffy cloth. respectively. Cleaning After the slides have soaked for 24 hours. Discard any slides that are stained. detach the coverslips and drop them into a beaker of water (Fig. Change the water three times.60) (for cleaning of coverslips. shaking the bowl vigorously each time.) Dirty slides Slides covered with immersion oil Take the oily slides one by one and rub them with newspaper to remove as much of the oil as possible. one by one. Rinsing Preferred method Remove the slides one by one from the weak detergent solution using forceps. stools).g. 61–80 and 81–100. 3.60 Removing coverslips from a slide for cleaning When slides have been used for infected specimens (e. Note: Detergents containing enzymes are excellent for removing blood films. Quick method Empty the bowl of weak detergent solution and fill with clean water. or try to clean them again. they should be placed in disinfectant solution before cleaning. one at a time. Rinse each slide separately under the tap. . then soak for 30 minutes in a bowl of water. the slides are numbered 1–20. Wiping. scratched or yellow or that have dull patches on them. Numbering In some laboratories the slides are numbered in advance in series of five packets with a diamond pencil.80 Manual of basic techniques for a health laboratory Wiping and drying Wipe the slides. urine. then drop into the bowl of weak detergent solution and leave to soak for 1 or 2 hours. Slides with coverslips Using the tip of a needle or forceps. 21–40. drying and wrapping up Follow the instructions given above for new slides. Leave to dry. with a soft. Soaking in detergent solution Prepare a bowl of cold or lukewarm water mixed with detergent. Examine each slide. (For example. Fig. Leave the slides to soak for 24 hours. 3. Wrapping up Divide the slides into piles of 10 or 20 and wrap each pile in a small sheet of paper. Rub each one with cotton wool dipped in the strong detergent solution.

soak the syringe for several hours in a bowl of 1 mmol/l hydrogen peroxide. shaking gently from time to time. Reusable syringe with blocked piston To loosen the piston. 1. all of which may harbour potentially infectious organisms. Blocked needles To remove the blockage. dry coverslips in a small Petri dish. Rinsing and soaking needles As soon as the needle has been used. rinse it while it is still attached to the syringe. may contain stools. 3). sputum. piston down.61). 2. 6. then remove it and leave it to soak in hot water. alternatively. use a nylon thread dipped in 50% acetic acid solution (reagent no. If possible. Drain the coverslips by tipping them out carefully on to a pad of gauze.3. Give a final rinse with demineralized water. Finally remove the needle and rinse the hub cavity. Dry in a hot-air oven at 60 °C. General laboratory procedures 81 Coverslips Used coverslips can be cleaned and reused. blood or urine. Leave the coverslips to soak for 2–3 hours. 3. if possible. . Leave for 10 minutes. remove the plunger from the used syringe and rinse both the barrel and the plunger.5. CSF. shaking gently. Fig. After loosening the piston. Put the coverslips into the beaker one by one. 7. you can use a stylet. insert the plunger and force the water through the needle. Make up the following solution in a large beaker: — 200 ml of water — 3 ml of detergent — 15 ml of bleach or 5 ml of a quaternary ammonium disinfectant (see pages 84 and 85). 4. Rinse out the beaker containing the coverslips with tap water four times. pus. Stand the syringe on its end. 5.61 Cleaning a blocked (reusable) syringe using acetic acid 3. 3) into the nozzle of the syringe with a fine Pasteur pipette (Fig.2 Cleaning non-disposable specimen containers Non-disposable containers. Keep clean. such as jars and bottles. Pipette 50% acetic acid solution (reagent no. Reusable syringes and needles As soon as a sample has been collected. choose one of the following methods: q q Soak for 2 hours in hot water (about 70 °C). use special coverslip forceps for taking them out. 3. Fill the barrel with water. 3.

2. Urine bottles Empty the bottles into the lavatory. 3. 10% solution (reagent no.82 Manual of basic techniques for a health laboratory Containers for stool specimens If the lavatory is not connected to a septic tank.62 Cleaning sputum pots by boiling in detergent — a 10% solution of household bleach (see page 84). Test-tubes containing blood specimens Test-tubes of fresh blood collected on the same day should be: — rinsed in cold water — left to soak in a detergent solution (see page 80). Clean with detergent and water. Fill them with either: Fig. Sputum pots and tubes containing pus or CSF specimens There are several possible methods. Clean the jars with detergent and water. 3. as described on page 80. Leave for 6 hours. Test-tubes of “old” blood kept for several days at room temperature may contain large numbers of microorganisms. Using formaldehyde solution or cresol Pour into each sputum pot either: — 10 ml of undiluted formaldehyde. 1 For further information. They should be: — filled with a 10% solution of household bleach (see page 84) — left for 12 hours and then — rinsed and cleaned. cresol or other disinfectants should not be added to the stools before disposal. or — a 5% solution of cresol (see page 83). Boiling in detergent Keep a large pan especially for this purpose. fill the jars containing stools with a 5% solution of cresol (see page 83) or a similar disinfectant.62). 1. as described on page 80. empty the contents into the sink or lavatory. . 28). or — 5 ml of 5% cresol (see page 83). If the lavatory is connected to a septic tank. Place the containers in the autoclave and sterilize for 30 minutes at 120 °C. After the containers have cooled.5. 3. Leave for 12 hours. Boil sputum pots for 30 minutes in water containing washing powder (60 g per litre of water) (Fig. Using an autoclave1 This is the best method. see section 3.5. Empty into the lavatory. Leave for 4 hours.

see section 3.5. acids or ethanol. Use 70% ethanol for metal bowls and 1% bleach (see page 84) for plastic ones. Lysol Lysol is an emulsion of 50% cresol in an aqueous solution of soap. but a 5% aqueous solution can be kept as a stock solution. then leave to soak in clean water for 12 hours. The temperature in the incubators should be recorded daily. Table 3. incubators must be cleaned at regular intervals (at least every fortnight) and also after spillage of any material. Change the water and clean the inside of the water-bath at least once a month or whenever it looks dirty.) Rinse the centrifuge buckets after use and remove any traces of blood.3.1 lists the disinfectants that are most commonly used in health laboratories. The incubator must maintain a constant average temperature of 35 °C (range 33– 37 °C). Use a thermometer to check the water temperature each time the water is changed as scale on the heating element may cause the thermostat to malfunction. Like all laboratory instruments. (Do not use bleach for metal bowls as it may cause corrosion. . Westergren tubes Rinse in water. Cresol can be replaced by phenol. Cresols emulsify well in soap solutions.3 Cleaning and maintenance of other laboratory equipment Centrifuges1 Clean the bowl of the centrifuge daily or after any spillage occurs. Dry completely (in an incubator at 37 °C. whether infectious or non-infectious. A crystal of thymol will help to prevent algal growth. but since phenol is a less powerful disinfectant the time of 1 For further information. according to the manufacturer’s instructions. The actual temperature must correspond to the thermostat setting when the instrument is used.3. 3. General laboratory procedures 83 3. Lubrication of the centrifuge should be carried out by a specialist. the carbon brushes may need replacing.3. Do not use washing powder.4 Disinfectants There are many disinfectants that have various different chemical actions on infective agents. they are less water-soluble than phenol. Check the wiring for fraying and loose connections at regular intervals. Water-baths If possible fill the water-bath with distilled water or rainwater to prevent deposits forming inside. etc. if possible).5. In carbon dioxide incubators used for microbial culture. Incubators Incubators are used for bacterial culture by laboratories working in microbiology. the concentration of carbon dioxide should be maintained at 5–10% and the humidity at 50–100%. If the centrifuge is sparking or running irregularly. Cresols Cresols may be solid or liquid.

household and industrial applications. 80% solution Iodine. 5% solution Cresol. exposure of material to phenol solution must be longer than for cresol. 5% solution Chloramine (5% available chlorine) Sodium hypochlorite (1% available chlorine) Laboratory instrumentsa Glassware a Sodium hypochlorite (0. undiluted solutions should contain 10% available chlorine.1 Commonly used disinfectants Intended object of disinfection Blood Disinfectant Recommended Minimum dilution for duration of disinfection (v/v) treatment 2:1 2:1 2:1 3:1 2:1 undiluted 1:1 1:1 undiluted undiluted undiluted undiluted undiluted undiluted undiluted undiluted undiluted undiluted undiluted undiluted undiluted undiluted undiluted 6h 6h 6h 6h 6h 6h 4h 4h 2 min 2 min 2 min 2 min 2 min 2 min 2 min 2 min 16 min 4h 4h 4h 4h 4h 12 h Stock preparation of disinfectant crystals or liquid powder crystals or liquid powder powder powder crystals or liquid crystals or liquid 50% cresol in soap solution 95% solution 5% solution pure pure pure powder solution powder 50% cresol in soap solution crystals or liquid powder powder 5%. 70% solution n-Propanol. 60% solution Chloramine (1% available chlorine) Quaternary ammonium compounds Drinkingwater Work benches Chloramine. Phenol and cresol solutions cause irritation of the skin and eyes. Hypochlorites cause irritation of the skin. 50% solution Ethanol. Hypochlorites are rapidly inactivated by particles of dust and organic materials and must be freshly prepared from stock solutions every day. 5% solution Calcium or sodium hypochlorite solution (1% available chlorine) Calcium hydroxide. They are used in a number of laboratory. Sodium and calcium hypochlorite Sodium and calcium hypochlorite solutions (household bleaches) are very strong disinfectants. slides or other glassware are discarded and for swabbing bench surfaces: 10 ml of concentrated hypochlorite solution in 990 ml of water (0. if neither sterilization nor highlevel disinfection by boiling is possible. 15% solution Cresol. 15% solution 5%. 1% solution Polyvidone iodine. Change the containers daily. 5% solution Calcium hypochlorite solution (1% available chlorine) Stool Cresol.84 Manual of basic techniques for a health laboratory Table 3.25% solution Cresol. Place the used glassware into the jars of hypochlorite solution and leave for at least 12 hours. Strong. 10%. 50% solution Cresol. For preparing working dilutions. 20% solution Chloramine (4% available chlorine) Urine Sputum Skin Cresol. 1% solution Isopropanol.1% available chlorine) Sodium hypochlorite (1% available chlorine) Chemical disinfection for skin-cutting and invasive instruments should be employed only as the last resort. 10%. the following dilutions are recommended: q For jars and containers in which used pipettes. and then only if the appropriate concentration of the chemical is available and if the instruments have been thoroughly cleaned to remove gross contamination before soaking in the chemical disinfectant. . eyes and lungs. 5% solution Cresol.1% available chlorine). Do not overfill the containers. 0.

although at a slower rate. releases chlorine as the active disinfectant agent. ethanol. syringes. Pasteur pipettes. General laboratory procedures 85 q For decontamination of blood spills and other specimens with a high protein content: 40 ml of concentrated hypochlorite solution in 360 ml of water (1% available chlorine). . like the hypochlorites. A solution of 1% available chlorine is obtained by dissolving 14 g of calcium hypochlorite in 1 litre of water. tubes. they are not toxic and are harmless to the skin. Some people are hypersensitive to iodine and suffer a rash on areas of skin that have been exposed to iodine solution. etc. Strong hypochlorite solutions are corrosive and can cause burns. viruses and mycobacteria. but not fungi. It is also used for water disinfection: chlorinated water has a concentration of 0.5 Sterilization Sterilization is defined as the destruction of all microorganisms in or about an object. They kill bacteria and some viruses. In the medical laboratory sterilization is achieved either by moist heat (autoclaving. Calcium hydroxide Calcium hydroxide solution is prepared from quicklime (calcium oxide) powder or granules dissolved in water (1 part : 3 parts w/v).3. n-propanol) are fast-acting. — to prepare the equipment used for bacteriological cultures (Petri dishes. They are not effective against spores. It decomposes at a slower rate than sodium hypochlorite. must be sterile).). Their sensitivity is much less when iodophores (polymer solutions that bind iodine) such as polyvidone iodine are used.5. At low temperatures iodine is more active than other disinfectants. viruses and fungi. tubes. Alcohols Alcohols (e. Chloramine Chloramine (tosylchloramide sodium) is a crystalline powder which. Materials are sterilized for three main purposes in the medical laboratory: — in preparation for taking specimens (needles. Calcium hydroxide solution is not suitable for disinfecting stools from patients with tuberculosis. Quaternary ammonium compounds Quaternary ammonium compounds (QUATS) are effective against vegetative bacteria and some fungi. fast-acting disinfectant with a wide range of action. Distilled water must therefore be used. but relatively expensive disinfectants that are usually used for skin disinfection. etc. isopropanol. Note that chlorinated water can interfere with laboratory tests. and eye shields to prevent splashing in the eyes. Calcium hypochlorite is available in its solid form as powder or granules. It kills bacteria. — to disinfect contaminated materials. Handle solutions of bleach carefully: wear rubber gloves to protect the hands. boiling) or by dry heat (hot-air oven. 3.g. flaming). many spores.05% chloramine. Iodine Iodine is an excellent.

Lid The lid covers and seals the boiler and is fitted with a rubber washer. Components of an autoclave (Fig. Temperature gauge or pressure gauge All gauges indicate the temperature in degrees Celsius (°C). Boiler A large deep cylinder in which the items to be sterilized are placed. 5. Air outlet valve A valve at the top of the boiler or on the lid that is used to let air out when the water is first heated. Lid clamps These clamps. Principle Water is heated in a closed container. 6.86 Manual of basic techniques for a health laboratory Sterilization by steam Using an autoclave Clinical samples and other contaminated waste materials are placed in a special autoclave bag or into a metal or plastic bucket for autoclaving. Use the autoclave sterilizing indicators to control the sterilizing cycle. Basket A big wire basket that holds the materials to be sterilized. Most types of microorganism. 9.63 Components of an autoclave 1: boiler. This produces saturated steam under pressure. Fig. 3. 4. 3: basket support. 2: basket. 3. some also have a second set of figures indicating the pressure. Safety valve A valve at the top of the boiler or on the lid that lets steam escape if the pressure becomes too high and so prevents an explosion. 2. 4: drainage tap. with a temperature of over 100 °C. 9: temperature gauge or pressure gauge. 7: air outlet valve. 7. 5: lid. seal the lid and prevent steam from escaping. including all bacteria (but not all viruses) are killed when apparatus is heated for 20 minutes at 120 °C in this steam under pressure. 8: safety valve. 8. 3. . Heating system The heating system may be built into the autoclave in the form of: — electric elements — gas burners — a paraffin oil stove. together with the rubber washer. 6: lid clamps. Drainage tap A tap fitted at the base of the boiler to drain off excess water.63) 1. Basket support A support in the bottom of the autoclave that holds the basket above the water level.

it should be kept away from flammable materials and chemicals. Alternatively they may be placed in autoclavable polyethylene bags. Fill the bottom of the autoclave with water (up to the basket support). 3. If gas or a paraffin oil stove is used for heating. etc. 3. The indicator papers turn black when the correct temperature is reached. The metal trays are placed uncovered in the autoclave. Otherwise. Put the basket containing the material to be sterilized in the autoclave together with sterilization indicator papers. Make sure that the water does not touch the basket. 3.65 Alternative method for autoclaving needles Fig.64). 3.64). 2.66 Autoclaving Pasteur pipettes . Petri dishes.64 Autoclaving syringes and needles Installation Autoclaves should be installed away from the main working area. 3. 3. or they are wrapped in gauze and placed in metal trays. Place a pad of non-absorbent cotton wool at the bottom of each tube to protect the tip of the needle. If necessary. Pasteur pipettes (Fig. should be wrapped in autoclavable polyethylene bags and tied with string.66) Pasteur pipettes should be placed in large tubes which are then plugged. Preparation of material for sterilization Reusable syringes Reusable syringes are placed in large glass test-tubes plugged with non-absorbent cotton wool (the pistons and barrels in separate tubes. Reusable needles Reusable needles should be placed separately in small test-tubes that are then plugged (see Fig.3. Sterilization procedure 1. 3. arrange the needles in metal trays with their points stuck into a folded gauze pad (Fig.65). General laboratory procedures 87 Fig. Fig. as they are noisy. Glassware Specimen tubes. drain off excess water by opening the drainage tap. Fig.

neither the pressure nor the temperature will be correct. dry the sterile equipment in an incubator at 37 °C. because if it is left for several hours without the outflow valve being opened. tubes of pus): 30 minutes at 120 °C. 2. Start timing at this point. Leave the autoclave to cool. but not too tightly. open the air outlet valve to equalize the pressures inside and outside the autoclave. Cleaning Wipe the inside of the autoclave daily or whenever spillages occur. Never open the lid before the pressure has dropped to normal. When the hissing sound stops. Never heat the autoclave too quickly to bring up the pressure once the outlet valve is closed. q q q Turning off the heat 1. Turn off the heat as soon as the required time is up. unscrew the lid clamps. Precautions Never touch the drainage tap. Bacteriological culture media: follow the instructions of the bacteriologist or the chief laboratory technician. Never leave the autoclave unattended while the pressure is rising. 120 °C) the heat must be regulated to maintain it. Tighten the lid clamps and reduce the heat slightly. This shows that all the air has been driven out of the autoclave.e. During sterilization make sure the lid is secured and no steam escapes as if it does. Screw down the lid clamps evenly and firmly. Begin heating the autoclave. When the temperature falls below 100 °C. Close the lid. outlet valve or safety valve of the autoclave while heating it under pressure. using steam under pressure. then carefully remove the basket of sterile equipment. q q q q q q Using a pressure cooker Pressure cookers are large saucepans designed to cook food very quickly. Watch the air outlet valve until a jet of steam appears. 4. 3. as you may be scalded with steam. Never leave the autoclave to cool for too long. tubes): 20 minutes at 120 °C. Watch the temperature gauge. Open the air outlet valve. making sure that the rubber washer is in its groove. 8. Take off the lid. . Sterilization times Materials for collecting specimens (reusable syringes and needles. 7. When the desired temperature is reached (i. They are used in some small laboratories to sterilize equipment used for specimen collection. Wait 3 or 4 minutes until the jet of steam is uniform and continuous. Containers of infected material (sputum pots. a vacuum forms. Close the air outlet valve. Reduce the heat until the needle on the dial remains at the temperature selected. if possible. If drops of water have formed. 6. 5.88 Manual of basic techniques for a health laboratory 3.

Sterilization by dry heat Using a hot-air oven This method should be used only for glass or metal articles (reusable syringes and needles. Put the water and material or object to be sterilized in the pressure cooker as described above. 2. if not available. Prepare the object to be sterilized in the same way as for the autoclave method. Leave the pressure cooker to cool (or cool it in cold water). 2. etc. Wait until the valve begins to whistle. which is fixed to the lid. Fit on the lid. pipettes. heat at 175 °C for 2 hours. 6. When it does. Leave the pressure cooker to cool (or cool it in cold water). Turn off the heat. Fill the pan with water (preferably demineralized) and heat over the stove. 5. Set the thermostat to 175 °C and switch on the oven. 3. 3. Open the valve so that air can enter. Leave the pressure cooker on moderate heat for 20 minutes. Fig. 4. Wait until the jet of steam is continuous. 3. forceps) should be put in when the water is boiling. 5. 3. Screw it down with its knob. Open the valve in the lid. The wrapped articles should be placed upright (never lay them flat.) when an autoclave is not available. Pull off the revolving valve so that air can enter. 2. Place the material or object to be sterilized in the basket (which is held above the water level by a support). continue heating at this temperature for a further 60 minutes. close the valve. Remove the lid. 3. Place the revolving valve (V1) on its shaft in the lid (Fig. Use a special boiling pan or. then lower the heat so that the valve keeps turning slowly. Fig.68). 4. reduce the heat. If there is a fan. Start heating the pressure cooker. As soon as a continuous jet of steam escapes from the valve. Warning: Never touch the safety valve. Sterilization by boiling This method should be used only where there is no alternative. Raise the lids of the metal boxes slightly and arrange them so that they face the back of the oven. Take out the sterilized material or object and leave the pressure cooker to dry.3. When the temperature reaches 175 °C. Cotton-wool plugs should not be too thick. Remove the lid. check that it is working. If the object to be sterilized is heavy or bulky or if it includes powders.68). Watch the thermometer. Start heating the pressure cooker on the stove. The valve soon begins to turn. Leave the articles to boil for 30 minutes. Take out the sterilized material or object and leave the pressure cooker to dry. Turn off the heat. which should be autoclaved (see page 86). otherwise the hot air cannot penetrate. General laboratory procedures 89 Pressure cooker with revolving valve 1. 3. 3. Glassware (reusable syringes) should be put in while the water is still cold.68 Components of a pressure cooker Fig. 6. a saucepan.67). Metal articles (reusable needles. Warning: Never touch the safety valve (V2 in Fig. letting a jet of steam escape. Leave the pressure cooker on moderate heat for 20 minutes. Pressure cooker with fixed valve 1. oils or petroleum jelly. 1.67 Sterilizing equipment using a pressure cooker . 3. It must not be used for culture media used in microbiology. Fill the bottom of the pressure cooker with water.

Add about 10 drops of ethanol and ignite. 3. Fig. 3. During flaming tilt the tray first one way. Place the articles in a metal tray. Cut a wide opening or vent (V) below the level of the grating.1 Disposal of specimens and contaminated material Any clinical material brought into the laboratory and any apparatus used to handle this material must be considered as infectious.6. 3. 3. Switch off the heat. 2.71). V: vent. Fig. 2. Fig.70) An old metal drum is suitable for this purpose. Find a removable lid (L) for the drum. 3. place all used stool and sputum boxes on the grating of the incinerator (Fig. To avoid laboratory accidents.8). Fix a strong metal grating (G) firmly about one-third of the way up the drum. then the other (Fig.2 Incineration of disposable materials Making an incinerator (Fig. 1.90 Manual of basic techniques for a health laboratory 4. 3.69 Sterilization by flaming To sterilize bacteriological loops.70 Components of an incinerator G: metal grating.6. Close the lids of the metal boxes. By flaming This method should be used only for metal articles such as forceps and scalpels. Open the oven door. 3. L: lid.69). Wait until the temperature falls to 40 °C. 3.71 Using an incinerator . 1. It is not suitable for general use. Remove the sterile object. heat them in the flame of a gas burner or spirit lamp until they are red hot. make sure that priority is given to correct handling and disposal of specimens and contaminated material (see section 3. Using an incinerator q At the end of each morning’s and each afternoon’s work. 3.6 Disposal of laboratory waste 3. 3.

and histological examination of biopsy material.74).7. It is advisable to strengthen the upper rim of the pit by lining it with bricks or stones.2 shows. Make a lid that fits tightly over the pit. . Fig. sticks. Place the specimen in the bottle or tube and seal hermetically (fixing the stopper with sticking-plaster.72). Double pack specimens. or more often if necessary. 3. If possible. for each type of specimen and each examination: — which container and preservative (where necessary) to use. Once a week. It should show: — the patient’s name (written in capital letters) and date of birth. — how much of the specimen to send. etc. Throw stool or sputum boxes and other infected material into the pit twice a day. General laboratory procedures 91 q Always keep the metal drum tightly closed (both lid and vent).3. Then place the sealed bottle in an aluminium tube with a screw cap. — the nature of the specimen. 3.72 Disposal of materials by burying 3. Fill the bottom of the drum with paper. q q The pit must be protected from animals. cover the refuse with a layer (about 10 cm thick) of dried leaves. Replace the lid immediately. 3.1 Packing specimens for dispatch Always observe the regulations in force in your country. see Fig. 3. Table 3. Incinerate once a week. For example. — how long the specimen will keep. birds and humans. — the date of collection of the specimen. Remove the lid. Wrap the request form around the metal tube (Fig. Light the fire and keep it burning until all the infected material has been reduced to ashes. except during incineration. Wedge it in the tube with absorbent cotton wool. instead of using dry leaves add a layer of quicklime (calcium oxide) once a week.6. wood shavings.7 Dispatch of specimens to a reference laboratory The peripheral laboratory sends specimens to reference laboratories or more specialized laboratories for examinations that cannot be carried out locally. q q 3. Check that the bottle is labelled with the patient’s name and the date of collection of the specimen. q q q 3.73). The ash produced is not dangerous and can be thrown on the refuse heap. A pit must never be constructed near a water source. culture of stools for detection of cholera vibrio. serological examinations for treponemal infection or typhoid.3 Burial of disposable materials Dig a pit 4–5 metres deep and 1–2 metres wide at a location where neither groundwater nor surface water can enter and where leaching of waste liquids into the groundwater cannot occur (Fig.

56) (see section 8.10) Serological tests for HIV and hepatitis B virus (see sections 11. including Vibrio cholerae (see section 5.9) Culture of all organisms. see sections 8.2. 22) Serological tests for syphilis (see section 11. etc.4) — 4 weeks — about 5 ml 2 weeks Keeps almost indefinitely Keeps almost indefinitely — .4) Container and preservative Amount of specimen to send — — — — — Preservation time 10 days 2 hours 24 hours 4 hours 24–48 hours 45-ml bottle containing 25 ml of cetylpyridinium bromide.4) Examination for vegetative forms of amoebae (see section 4.7 and 11. 28) 10-ml tube containing thiomersal– iodine–formaldehyde (TIF) fixative fixative (reagent no.3.4.. 0. 56) Sterile tube Swab of pus 1 ml 5 ml 10 ml 24 hours 2 hours 12 hours 3 days Erythrocyte and leukocyte cell EDTA dipotassium salt. 10% solution counts (see sections 9.2 Dispatch of specimens to a reference laboratoryof basic techniques for a health laboratory Type of specimen Sputum Type of laboratory examination Culture of tubercle bacilli (see section 5. 44) 5 ml 24 hours Stools Culture of all organisms. 58) or polyvinyl alcohol (reagent no. modified (reagent no. except Vibrio cholerae Examination for parasite ova.2. larvae and cysts (see section 4.4) Culture of other organisms Throat swabs Culture of diphtheria bacilli (see section 5.5) Bacteriological culture (see section 5) Sterile bottle Sterile bottle 2–4 ml Special bottle of Stuart transport medium.92 Manual Table 3. 10% solution (reagent no. modified (reagent no.8) Tests for glucose (see section 10.5) Urethral pus Pus from other sources Blood (see sections 9–11) Culture of gonococcus (see section 5. 17) Buffered glycerol saline (reagent no.6% solution No preservative Tube containing coagulated serum Cotton-wool swab CSF (see section 8) Culture of meningococcus Special bottle containing Stuart transport medium.1) Other biochemical tests: q bilirubin q cholesterol q serum iron q serum lipids q proteins q liver function q uraemia Enzyme estimations: amylase phosphatase q transaminases q Sterile tube without anticoagulant. chloride. send serum or dried drops of blood as appropriate Send successive specimens of serum: taken at the onset of the disease q taken after 2–4 weeks (to detect q increase in antibodies) q 5 ml 24 hours 5 mg of sodium fluoride Bottle without anticoagulant (send serum) 5 ml 10 ml 2 hours 48 hours Bottle without anticoagulant 5 ml 2 hours Culture Special sterile flask containing 50 ml of broth raised to 37 °C as quickly as possible after adding the specimen Cary–Blair transport medium (reagent no.2) Sterile airtight bottle sent in a vacuum flask filled with water at 37 °C 2 ml 2 ml 12 hours 2 hours 2 hours Culture of other organisms Chemical tests (for glucose.3. protein. 14) 30-ml bottles containing 15 ml of formaldehyde.4 and 8.5 and 9.6) (reagent no.

10% solution (reagent no.65 Date of collection of specimen: 9. General laboratory procedures 93 Table 3.2. Wedge the tube in tightly with non-absorbent cotton wool.3. Mwanza Name of patie nt: J. nails. 66) q Paper envelope or screw-capped bottle (do not use tubes with rubber stoppers or plugged with cotton-wool) — At least a week (sometimes longer) Name and address of health facility requesting exam ination: Outpatients’ Clinic.2. Maternity Hospi tal.7. 3.01 W HO 99 07 2 Fig.7) Clean dry bottle Bottle containing 8 drops of formaldehyde. 28) For concentration: 2 ml of household bleach and 1 ml of hydrochloric acid Sterile bottle Sterile bottle 2 hours 2 days Schistosome eggs (see section 7.2) Examination for fungi (mycoses) (see sections 6.8. FRAGILE and. etc.2. depending on number of tests to be performed 30 ml 30 ml Preservation time 2 hours Clean dry bottle (sealed) Urinary deposit (see section 7.4–7. 27) q Zenker fixative (reagent no.8) Bacteriological culture (see section 5) Pregnancy test (see section 11. Label the outside of the box: URGENT. — the examinations required (with the physician’s diagnosis. Place the metal tube in a strong cardboard or wooden box for dispatch.73 Packing specimens for transport — the address of the health facility where the specimen was collected.75).1 and 6. 3.) Type of specimen Urine (see section 7) Type of laboratory examination Biochemical tests (for glucose.. . where appropriate). acetone. Smith Sex: F Nature of sp ecimen: Ur ine Name of patient: J. INFECTIOUS MATERIAL (Fig.5) Biopsy tissue (from an organ) Hair. if appropriate.3) 100 ml Keeps almost indefinitely 1 hour 12–24 hours (or 4 days in refrigerator) — 20 ml 20 ml (first urine of day) — The following fixatives are used: formaldehyde saline (reagent no.7. cutaneous tissue Histological examination (see section 3.2. see sections 7. It should also be signed by the physician. Smith Sex: F Date of birth: 3.6) Container and preservative Amount of specimen to send 20–50 ml.2 (cont. protein.

0 ired 1 : WHO 99073 Se Na x: F Da ture Exa te of of spe min coll cim atio ectio en: ns n: Urin req 9.7.75 Label the box containing the specimen .74 Wrap the request form around the metal tube containing the specimen Sex Natu : F Date re of s E xam of coll pecim inati ecti en: U ons on: 9 rine requ . 3. 3.7 e uire .94 Manual of basic techniques for a health laboratory Fig.01 d: W HO 99 07 4 Fig.

The area of the specimen. The laboratory technician must be able to fix the biopsy specimen and to ensure that it is properly dispatched and arrives at the pathology laboratory in a good state of preservation. It is examined under the microscope after a thin section has been cut and treated with a special stain. Technique Amount of fixative The volume of fixative required is about 50 times the volume of the biopsy tissue. This type of examination is called histopathology and can be most important. Just before use. etc.3). autolysis. Histopathology The cells of biopsy specimens from tissues and organs can be studied under the microscope.5 ¥ 1 1¥1 2¥1 2¥2 Amount of fixative (ml) 6–10 10–15 20–25 30–40 90 . The most suitable type of bottle for biopsy specimens is a plastic-capped bottle with a wide mouth (pill bottle). can vary and this is what determines the amount of fixative to be used (see Table 3. the physician removes a piece of tissue with forceps or a special scalpel. 45-ml. General laboratory procedures 95 3. add 5 ml of glacial acetic acid per 100 ml of Zenker solution. This piece of tissue is called a biopsy specimen.3 Calculating the amount of fixative to use for biopsy material Dimensions of specimen (cm) 0. Fixation of biopsy specimens The piece of tissue is immersed in a fixative fluid. particularly for the diagnosis of cancer.2 Fixation and dispatch of biopsy specimens for histopathological examination Biopsy specimens To diagnose certain diseases of the organs.5 0. This procedure should preserve the tissue in a state as close to the living state as possible. 27). fixation is difficult or impossible).7. 66). Such bottles are obtainable in 60-ml. — Zenker fixative (reagent no. Biopsy tissue is normally 3–5 mm thick (if it is thicker. by protecting it against bacterial action. 30-ml and 15-ml sizes. Fixatives Fixatives that are simple to prepare are: — formaldehyde saline (reagent no. shrinkage. however. Table 3.5 ¥ 0.3.

write on it the name of the patient. Always consider any laboratory specimen as potentially infectious and handle it carefully.). the specimen can be left in the liquid for at least a week before it is cut and stained. Then use a stiff brush or sheet of cardboard to sweep it into a disposable specimen container. Place all specimens safely on a bench or in a rack to prevent spillage or breakage. parasites.) Once filled.6. Do not contaminate yourself or the work areas with any specimen. With the two fixatives mentioned above. Fixation time This will vary according to the fixative used.4). hepatitis B virus. (Sharps containers can be made from plastic bottles with a screw top in which a hole is made. Wear protective clothing and remove it before leaving the laboratory. The laboratory should have a first-aid box (see section 3.5. visitors should be restricted. Never leave it until later.2 and 3. which may damage the tissue). containers should be autoclaved or soaked in disinfectant before burning or burying in a deep pit (see sections 3.g. the nature of the specimen and the date of collection. Dispose of used needles and lancets safely in a “sharps” container. wear protective gloves. First pour the fixative into the bottle. together with the request form (see section 3. Cover all cuts with an impervious dressing (plaster). Then place the tube and the request form in a small wooden or cardboard box and dispatch immediately. Take great care when collecting and processing blood samples as they may harbour infective agents (e. Then pick up the biopsy specimen on a piece of stiff paper (do not use forceps.8. q q q q q q q q q q q q q .1). Using a lead pencil. etc. Cover any spilled material or broken culture tubes with a cloth soaked in disinfectant (see section 3. but a long transit period will not result in the deterioration of specimens.2) and at least one staff member trained in first aid. Dispatch of biopsy specimens Secure the cap or stopper of the bottle with adhesive plaster. Fixed material should be dispatched to the pathology laboratory without delay.7. Drop the specimen into the bottle. Place the slip of paper in the bottle with the fixative.3). No food or drink should be consumed in the laboratory.4) and leave for 30 min.6. 3.96 Manual of basic techniques for a health laboratory Preparation It is essential to act quickly on receipt of a biopsy specimen. Labelling Cut out a small rectangle (about 3 cm ¥ 1 cm) of stiff paper. Place the bottle in an aluminium tube with a screw cap. The laboratory should be a work area only. Do not pipette blood or other body fluids or any reagents by mouth. At the end of the day swab the benches with a cloth soaked in disinfectant (see section 3.5.8 Safety in the laboratory q Each laboratory should have a written manual of safe laboratory practices which should be followed at all times.

stirring the mixture after each drop. Never place a bottle of ether on a workbench where there is an open flame. Propane and butane gas burners When lighting a gas burner. Heat the middle of the tube. sterilized and used again (see sections 3. Warning: Ether will ignite at a distance of several metres from a flame. watching the level of the liquid. Disposable containers must not be reused.1. Ordinary glass will break. Pipette slowly.1 Precautions to prevent accidents Handling acids and alkalis Diluting concentrated sulfuric acid with water Always add the concentrated sulfuric acid to the water drop by drop. sputum). General laboratory procedures 97 q Wash your hands well after handling infective material and before leaving the laboratory. Do this in a sink whenever possible.5. When you take a bottle out make sure your hand is dry and hold the bottle firmly upright. use a pipette with a rubber safety bulb attached. towards an empty work space or a sink. Bottles of acids and alkalis Keep bottles of acids and alkalis on the lower shelves of the cupboards. Flammable liquids Only small quantities of flammable liquids such as ether. The mouth of the tube should be facing away from you and any other person. always light the match and hold it to the burner before turning on the gas tap. acetone. benzene and toluene should be kept in the laboratory. shaking gently. Pipetting Use small measuring cylinders for measuring acids and alkalis. Heat-resistant glass Only heat-resistant glassware and porcelain receptacles can be heated over a Bunsen flame. Do not keep acids and alkalis in bottles with ground glass stoppers (they may get stuck). 3. Turn off the main valves of all bottles of butane gas every evening. If more accurate measurement is required. Replace the rubber connecting pipes once a year. — in glass jars and bottles that can be cleaned.5. the liquid inside might sputter. Specimens may be disposed of: — in cardboard cartons or plastic pots that can be destroyed (stools.5). .3.8. ethanol. Never pour the water into the sulfuric acid because of the danger of splashing due to the explosive evaporation of water while mixing.2 and 3. 3. Heating glassware and liquids Test-tubes Never heat the bottom of a test-tube.5.

8. saturated solution (reagent no. hydrochloric acid.98 Manual of basic techniques for a health laboratory 3. In all cases: Wash the affected area immediately with large quantities of water. 12) (in an eyedrop bottle) Acetic acid. hot liquids. The solutions should be kept in plastic bottles. First-aid box The first-aid box should contain the following: q q q q q q q q q An instruction sheet giving general guidance Individually wrapped sterile adhesive dressings in a variety of sizes Sterile eye-pads with bandages for attachment Triangular bandages Sterile dressings for serious wounds A selection of sterile unmedicated dressings for minor wounds Safety-pins A bottle containing eye drops A first-aid manual. Bathe the affected skin with cotton wool soaked in a 5% solution of sodium carbonate. The above items should be readily available in the laboratory. 5% solution (reagent no. It is therefore essential to take immediate action in the event of an accident. 50) (in an eyedrop bottle) Boric acid. swallowing. .2 First aid in laboratory accidents Accidents in the medical laboratory may have various causes: q q q q Acids or alkalis: splashes on the skin or in the eyes. First-aid equipment q q q q q q q First-aid box (see below) Sodium carbonate. flammable liquids. 52) Sodium bicarbonate. acetic acid and trichloroacetic acid can cause corrosive injuries. Corrosive injuries from acids Acids such as nitric acid. Heat: naked flames. 1) Cotton wool and gauze Mercurochrome and tincture of iodine. explosions. 2% solution (reagent no. Toxic substances. etc. The contents of the first-aid box should be replenished immediately after use and inspected regularly to ensure that they remain in satisfactory condition. sulfuric acid. electric shocks. 5% solution (reagent no. Injuries involving infectious material. Acid splashes on the skin q q Wash the affected area thoroughly and repeatedly with large quantities of water. chromic acid. They must not be kept in a locked cupboard.

76). squirt the water into the corner of the eye near the nose.3. wash the eye with running water from a tap (Fig. General laboratory procedures 99 Acid splashes in the eye q Wash the eye immediately with large quantities of water sprayed from a polyethylene bottle (or rubber bulb) for 15 min (Fig. potassium hydroxide and ammonium hydroxide can also cause corrosive injuries. If neither of these is available. . q q Fig. 3. two egg whites mixed with 500 ml of water may be given). never by mouth. put 4 drops of a 2% solution of sodium bicarbonate into the eye.77 Rinsing the eye under the tap Swallowing acids If acid is accidentally swallowed: q q Send for a physician. Such injuries may be more serious than those caused by acids. Send for a physician. then — bathe with a 2% solution of sodium bicarbonate. Ask the patient to close the eye that is not affected. the patient should drink ordinary water. 3. 3. Make the patient drink some milk immediately (alternatively. 3. Continue to apply bicarbonate solution to the eye until the physician arrives. Give the patient three or four glasses of ordinary water.77). In all cases: Wash the affected area immediately with large quantities of water. After washing. Corrosive injuries from alkalis Alkalis such as sodium hydroxide. Make the patient gargle with the milk. If the lips and tongue are burned by the acid: — rinse thoroughly with water.76 Rinsing the eye using a polyethylene bottle Fig. q q q Note: Always pipette acids using a rubber safety bulb. Alternatively.

100 Manual of basic techniques for a health laboratory Alkali splashes on the skin q q Wash the affected area thoroughly and repeatedly with water. Cover the patient if he or she is cold. Lay the victim on the ground. Inform the physician on duty at the casualty department immediately. Alternatively. Alkali splashes in the eye q Wash the eye immediately with large quantities of water sprayed from a polyethylene bottle (or rubber bulb). Do not remove any clothing.77). burns caused by hot glassware or a Bunsen flame).g. Bathe the affected skin with cotton wool soaked in a 5% solution of acetic acid (or undiluted vinegar or lemon juice). q q q . Immediately make the patient drink a 5% solution of acetic acid (or lemon juice or vinegar diluted 1 part vinegar to 3 parts water). If the lips and tongue are burned by the alkali: — rinse throughly with water. specifying the toxic substance involved. 3. 3. wash the eye with running water from a tap (see Fig.g. q Severe burns q If the victim is on fire (e. Make the patient gargle with the same acid solution. q q q Poisoning This can be caused by: — inhaling toxic vapours or gases (e. Burns caused by heat These fall into two categories: q Severe burns (e. squirt the water into the corner of the eye near the nose (see Fig. chloroform) — accidental swallowing of a poisonous solution. In all cases: q q Send for a physician or qualified nurse. Minor burns (e.g. bathe it with a saturated solution of boric acid. q q Swallowing alkalis If alkali is accidentally swallowed: q q Send for a physician. roll him or her in a blanket to extinguish the flames. Give the patient three or four glasses of ordinary water. burns caused when burning ether or boiling water is spilled over the victim). Place the victim in the open air while waiting for the physician or nurse.g. specifying that a patient with severe burns will have to be moved. Send for a physician. Do not apply any treatment to the burns: this must be left to the physician. After washing the eye with water. then — bathe with a 5% solution of acetic acid. Continue to apply boric acid solution to the eye until the physician arrives.76). splashed with burning ether or other flammable solvents).

mercurochrome or tincture of iodine). Wash the whole area thoroughly with soapy water. etc. Apply a dry gauze dressing loosely. q q q Before doing anything else. Continue to press on the covered wound while waiting for the physician or nurse. stop the bleeding by pressing down on it with a sterile compress. In case of a cardiac arrest.9 Quality assurance in the laboratory Quality assurance in the laboratory includes all aspects of the analytical work. The prime objective of quality assurance is to ensure that the laboratory provides results that are correct and relevant to the clinical situation of the patient. bacterial cultures. The symptoms are fainting. particularly with wet hands.g. refer the patient to a physician. try to stop the bleeding by pressing down on it with a sterile compress and send for a physician or qualified nurse. . cut off the electricity at the main fuse. q q q q Electric shocks Alternating electric current (120 V or 220 V) is usually used in the laboratory. bacterial cultures. q q q Note: Never tear off blisters that form over burns! Injuries caused by broken glass Clean glass q Disinfect the skin in the normal way (using. Refer the patient to the casualty department. massage the heart externally if necessary and begin giving artificial respiration. 3. If the cut bleeds heavily with the blood spurting out at intervals.1.: q Check whether the cut is bleeding. Electric shocks may occur when faulty equipment is being handled. asphyxia and cardiac arrest. Refer the victim to the physician if the material involved is known to be infective (e. If the cut is minor.3. Bathe the area again with tincture of iodine. Bathe the whole area (the edges of the cut and inside the cut) with tincture of iodine or a surgical antiseptic (see Table 3. pus. cover it with a sterile adhesive dressing (ready-made type). If the cut bleeds profusely. page 84). (He or she will decide whether a tourniquet should be applied. Apply mercurochrome or tincture of iodine to the burn.) q q q q Glass containing infected material Glassware containing stools. squeeze hard to make it bleed for several minutes. General laboratory procedures 101 Minor burns q Plunge the affected part into cold water or a mixture of ice and water to soothe the pain. If the burn becomes infected or does not heal. pus). if not. from correct identification and preparation of the patient to ensuring that the laboratory result goes back to the doctor. for example. Send for a physician.

2.g. it should always be collected at the appropriate time. are outlined in the relevant sections of this manual.102 Manual of basic techniques for a health laboratory The stages at which quality assurance should be applied include: — preparing the patient — collecting the specimen — handling and dispatch of the specimen (see sections 2.1 Specimen collection The appropriate collection of specimens is of utmost importance if the laboratory results are to be relevant to the clinical situation of a patient.6. the following factors should be considered: — the physiological state of the patient (e. — the appropriate preparation of patients for specimen collection (e.6. Specific aspects of specimen collection. blood for the measurement of glucose and lipids should be taken in the morning from a patient who has fasted for 12 hours. — the appropriate site for specimen collection (e.5) — reporting results (see section 2. the concentration of glucose is different in arterial and venous blood). For example.g. while urine for the diagnosis of schistosomiasis and other conditions should be collected as a “terminal” urine specimen (see section 7. sputum specimens for the detection of tubercle bacilli should be collected in the early morning. 3.g. .7) — control of methods and reagents (see individual methods) — calibration of equipment (see section 2. including those for the detection of infective organisms (bacteria and parasites). the reference ranges of certain indicators vary with age and sex).9. When material is collected for the purpose of monitoring and control of treatment of patients.g. Random collection should be limited to emergency situations. — the appropriate tools for specimen collection (e. because their concentrations are elevated after a meal).2).8). blood for cell counting should be collected in tubes containing EDTA dipotassium salt to avoid plasma coagulation and platelet aggregation). To ensure that the most useful specimen is obtained.1 and 3.

Parasitology 103 Part II .4.

104 Manual of basic techniques for a health laboratory .

1 Introduction A parasite is an organism that lives in or on another living organism of a different species. A parasite that lives in its host. Table 4. A parasite such as a tick that lives on its host is called an ectoparasite. They are an important cause of diarrhoea (see Table 4. Strongyloides stercoralis Trichuris trichiura Hymenolepis nana Heterophyes heterophyes Non-infectious Malabsorption syndromes Tropical sprue Crohn disease Whipple disease Intoxications Food poisoning Chemicals Drugs Inborn errors of metabolism Metabolic disorders Carbohydrate intolerance Gluten enteropathy Adrenal disease 105 Specific cause . Viruses Helminths Rotaviruses Fasciolopsis spp. this can be determined by examination of a stool specimen. Parasitology 105 4. Plasmodium spp.1 Common causes of diarrhoeal disease Type of cause Infectious Protozoa Amoebae Giardia spp. The organism from which the parasite takes its food is called the host.4. If acute diarrhoea is caused by parasitic infection. Bacteria Salmonella spp. Many diseases are caused by infection with parasites. Shigella spp. Parasitology 4. Escherichia coli Vibrio cholerae Staphylococcus spp. which is a major health problem in developing countries. such as a hookworm or an amoeba. is called an endoparasite. Balantidium coli Isospora belli Cryptosporidium spp. Campylobacter spp.1).

2 Routes of transmission of intestinal parasites Scientific name Helminths Ancylostoma duodenale Ascaris lumbricoides Clonorchis sinensis Dicrocoelium dendriticum. eating unwashed raw vegetables and salads. D. contact with infected persons with dirty hands. ascaris Chinese liver fluke Lancet fluke Fish tapeworm Dog tapeworm Pinworm. autoinfection Eating undercooked infected meat Walking barefoot on ground contaminated by stools. playing on contaminated ground (children) Eating unwashed raw vegetables and salads. playing on contaminated ground (children) Eating undercooked infected freshwater crabs Bathing in streams or ponds contaminated by infected stools or urine Bathing in streams or ponds contaminated by infected stools or urine Bathing in streams or ponds contaminated by infected stools or urine Bathing in streams or ponds contaminated by infected stools or urine Eating undercooked infected meat Eating undercooked infected meat Eating unwashed raw vegetables. contact with infected persons with dirty hands. particularly in the laboratory. hospes Diphyllobothrium latum Dipylidium caninum Enterobius vermicularis Hookworm Roundworm. They can then give advice on hygiene to members of the community and avoid infection themselves. playing on ground contaminated by stools (children) Eating undercooked infected meat Swallowing infected ants (in unwashed salad or while playing in grass (children)) Eating raw or undercooked freshwater fish Swallowing dog fleas (children) Walking barefoot on ground contaminated by stools. failure to observe safety regulations relating to cleanliness in the laboratory Common name How infection is contracted Fasciola gigantica Fasciola hepatica Fasciolopsis buski Heterophyes heterophyes Hymenolepis diminuta Hymenolepis nana Metagonimus yokogawai Necator americanus Paragonimus westermani Schistosoma haematobium Schistosoma intercalatum Schistosoma japonicum Schistosoma mansoni Taenia saginata Taenia solium: — adults — larval form (cysticercus) Trichostrongylus spp. contact with infected pigs (on farms) Drinking contaminated water. failure to observe safety regulations relating to cleanliness in the laboratory Eating unwashed salads Eating unwashed salads Eating unwashed salads Eating undercooked infected meat Swallowing rat fleas Eating contaminated vegetables. contact with infected persons with dirty hands.106 Manual of basic techniques for a health laboratory It is useful for laboratory technicians to understand the ways in which people can become infected by intestinal parasites (see Table 4. autoinfection. eating unwashed raw vegetables and salads. autoinfection — Whipworm — — Eating unwashed salads (Asia) Eating unwashed raw vegetables and salads Eating unwashed vegetables. Trichuris trichiura Protozoa Balantidium coli Entamoeba histolytica Giant liver fluke Liver fluke Giant intestinal fluke Dwarf fluke Rat tapeworm Dwarf tapeworm Japanese fluke Hookworm Oriental lung fluke Schistosome (vesical) Schistosome (rectal) Schistosome (Asiatic or Oriental) Schistosome (intestinal) Beef tapeworm Pork tapeworm Giardia intestinalis — . failure to observe safety regulations relating to cleanliness in the laboratory Drinking contaminated water.2). threadworm Walking barefoot on ground contaminated by stools. Table 4.

4. Colour The colour can be described as: — black (occult blood) — brown.2.2 Examination of stool specimens for parasites 4. Make sure that any adult worms or segments passed are included. — to detect erythrocytes. see section 5.2 Visual examination Faecal samples are best described by their colour. usually seen as streaks of red or white. schistosomiasis).1 Collection of specimens Collect approximately 100 g of faeces in a clean. Consistency (Fig. — to detect ova and cysts present in moderate numbers. Parasitology 107 4.g. For collection of stool specimens for bacteriological examination (e. as they may contain motile amoebae (which die quickly). Never examine stool specimens without first putting on gloves.1 Assessing the consistency of faecal specimens . in a bedpan). Select unformed or liquid faeces when using direct microscopy for detection of trophozoites. 4. cellular debris or excess fat.g. consistency and presence or absence of macroscopic blood or exudate.g. Never accept stool specimens mixed with urine (e. examine the liquid stools and those containing mucus or blood first.2.4.4. Always examine stool specimens within 1–4 hours after collection. Also perform a direct examination of any external blood or mucus.2.3 Microscopic examination Direct microscopic examination of faeces in saline or iodine suspension is useful for the following reasons: — to detect motile trophozoites. dry container without preservatives. A screw-top container is most suitable (see section 2.9. 4.5).1) The consistency can be described as: — formed (normal shape) — soft formed — unformed or liquid (watery). pale yellow (fat) — white (obstructive jaundice). If several specimens are received at the same time. ulcerative colitis. 4. The presence of external blood or mucus. Formed stools rarely contain motile trophozoites. Precautions q q q q Never leave stool specimens exposed to the air in containers without lids. for culture of cholera and other bacteria that cause dysentery). Blood may be present in certain medical conditions (e. Fig.5. should be noted.

Using the applicator or wire loop. 37) Acetic acid. Dilute the mixture with four volumes of distilled water and stir.2 Materials and reagents for direct microscopic examination of faeces for parasites Fig. take a small portion (about 2–3 mm diameter) of the stool. 4. Examine the preparations under the microscope. Mix the sample with the drop of sodium chloride solution on the slide. 7. Prepare a 1:1 mixture of Lugol iodine solution and acetic acid solution (diluted as above).5 to avoid the formation of air bubbles). 6. Using an applicator or wire loop. 4. 2. 3. 50% solution (reagent no. For the saline preparation use the ¥ 10 and ¥ 40 objectives and a ¥ 5 eyepiece. Discard the applicator (or flame the wire loop) after use. 4.108 Manual of basic techniques for a health laboratory Materials and reagents (Fig.45 mm. take the portion from the centre of the sample (Fig. 4.85% solution (reagent no. As the eggs and cysts are Fig.5% solution (reagent no. 5. Put: — one drop of sodium chloride solution warmed to 37 °C in the middle of the left half of the slide. 8.3 Adding a drop of saline and a drop of iodine suspension to the slide . and — one drop of the iodine–acetic acid solution in the middle of the right half of the slide (Fig. (a) If the stools are formed.3). diluted 1:1 with distilled water Methylene blue solution (reagent no. 24). 39) Eosin. Method 1. 3). take a second portion of stool from the specimen as described above and mix it with the drop of iodine–acetic acid solution. 53) Lugol iodine. 4. 4. Take a dry microscope slide and label it with the name or number of the patient. 0.4) and from the surface to look for parasite eggs. (b) If the stools contain mucus or are liquid. 4.2) q q q q q q q q q q Microscope Microscope slides Coverslips Wooden applicators or wire loops (0. take the portion from the mucus on the surface or from the surface of the liquid to look for amoebae. nickel–chromium alloy wire) Grease pencils Sodium chloride. 2% solution in saline (reagent no. 0. Place a coverslip over each drop (apply the coverslips as shown in Fig.

The nucleus is clearly stained but it may be difficult to distinguish between trophozoite and cystic forms. Fig. 4. allow a drop of methylene blue solution to run under the coverslip over the saline preparation (Fig. Examine the first preparation with the ¥ 10 objective.5 Technique for applying a coverslip to avoid the formation of air bubbles Fig. 28). 37). Focus on the edge of one coverslip using the ¥ 10 objective and examine the whole area under each coverslip for the presence of ova and larvae of Strongyloides stercoralis. 4. 4. Lugol iodine–acetic acid solution causes the trophozoite forms to become nonmotile.2. 11. — thiomersal–iodine–formaldehyde (TIF) fixative (reagent no.4. which remain colourless and are thus easily recognized.5% solution (reagent no. Parasitology 109 colourless.4 Sampling a faecal specimen for parasite eggs 4. 10% solution (reagent no. reduce the amount of light using the condenser aperture or lower the condenser to increase the contrast. 0. 4. This will stain the nuclei of any cells present and distinguish the lobed nuclei of polymorphs from the large single nuclei of mucosal cells.7 Staining the saline preparation with methylene blue Fig. 10. the whole field becomes stained except for the protozoa (particularly amoebae). In such cases a preservative should be added to the specimens before they are dispatched for examination.6 Examining the area under the coverslip for parasites . Using a fine Pasteur pipette. 4. — polyvinyl alcohol (PVA) fixative (reagent no. Then switch to the ¥ 40 objective and again examine the whole area of the coverslip over the saline for motile trophozoites and the area of the coverslip over the iodine for cysts.7). Fig. 58). for wet mounting. 44). If a drop of eosin solution is added. 9. — Lugol iodine.6. 4. starting at the top lefthand corner as indicated in Fig. The following preservatives are used: — formaldehyde. for wet mounting.4 Dispatch of stools for detection of parasites Stools may be sent to a specialized laboratory for the identification of rare parasites that are difficult to recognize.

4. including vegetative forms of amoebae (those of flagellates deteriorate slightly). On a slide 1.8). . 4. 4. To examine for amoebae and flagellates.7 ml of TIF fixative and 0. Add approximately 2 ml (2 cm3) of stool and crush well with a glass rod. Crush the stool thoroughly with a glass rod (Fig.9). Fig. The above-mentioned mixture preserves all forms of parasites indefinitely. Leave to dry for 12 hours (preferably at 37 °C). Using polyvinyl alcohol fixative Fig. carefully spread the specimen over about half of the slide (Fig. Using a glass rod. Fig. Just before dispatch.9 Crushing the faecal specimen with a glass rod 3. 4.10). 3. 2. mix 4. Add enough fresh stools to fill the last quarter of the bottle. It does not preserve vegetative forms of protozoa. which are destroyed after a few days. 2. Add three drops of PVA fixative to the stool. 2.3 ml of Lugol iodine solution in a tube or a small bottle. Pour about 30 ml of PVA fixative into a 40-ml bottle.10 Spreading a faecal specimen on a slide 1 Also known as thimerosal and mercurothiolate.8 Preserving a faecal specimen in formaldehyde solution In a bottle 1. 2. place a small portion of the stool on one end of the slide. 4. Mix thoroughly with a glass rod.110 Manual of basic techniques for a health laboratory Using 10% formaldehyde solution 1. Formaldehyde solution preserves eggs and cysts of parasites indefinitely if the specimen container is tightly closed. Prepare a mixture containing about one part of stool to three parts of formaldehyde solution (Fig. Specimens preserved in this way can be kept for about 3 months. PVA fixative preserves all forms of parasites indefinitely. 4. They can be stained on arrival at the specialized laboratory. Using thiomersal1–iodine–formaldehyde fixative 1.

4. . others are harmless. May cause dysentery or abscesses Non-pathogenic. 4.3 Intestinal protozoa Protozoa are microorganisms consisting of a single cell. yeast cells.3). F: flagellate.12): — size — cytoplasm — pseudopodia — nuclei — ectoplasm — endoplasm — vacuoles — inclusion bodies containing erythrocytes. Dientamoeba fragilis Flagellates Giardia intestinalis Trichomonas hominis Chilomastix mesnili Ciliates Balantidium coli Pathogenic Pathogenic Non-pathogenic Non-pathogenic Only amoeba that is commonly pathogenic to humans. — or because they have rapidly moving flagella (long whip-like threads) or cilia (numerous short hairs). Intestinal protozoa may be found in stools in their motile form (trophozoites) or as cysts. Iodamoeba butschlii.11): — either because of slow movements of the cell (amoebae). Trophozoites are chiefly found in: — watery stools — stools containing mucus — soft formed stools. bacteria. it is enough to be able to distinguish these species from Entamoeba histolytica Pathogenicity 4.3 Pathogenicity of intestinal protozoa Species Amoebae Entamoeba histolytica Entamoeba coli Entamoeba hartmanni. C: ciliate. etc. 4. debris. Some intestinal protozoa are pathogenic (see Table 4. Fig.4.11 Motile forms of protozoa A: amoeba. Differentiation is difficult but not really necessary. Parasitology 111 Table 4. 4. Endolimax nanus.1 Identification of motile forms (trophozoites) The trophozoites of protozoa are motile (Fig.3. — nuclear membrane (chromatin) — nuclear karyosome — flagella — undulating membrane. All these protozoa are found throughout the world. The following features are useful for the identification of motile forms of intestinal protozoa (Fig. but very common Non-pathogenic.

112 Manual of basic techniques for a health laboratory Cytoplasm Nucleus Pseudopodium Ectoplasm Endoplasm Vacuole Inclusion bodies: (a) containing erythrocytes (b) containing bacteria. 4. yeast cells and/or cell debris Nuclear membrane (chromatin) Nuclear karyosome Flagellum Undulating membrane Fig.12 Features for the identification of motile forms of protozoa .

yeast cells. but when stained with iodine solution clearly seen to have a regular membrane and a small dense central karyosome (a black dot). Nucleus: not visible in the motile form. 4. quite different from the fine granular texture of the endoplasm (greyish with yellowish-green streaks).14) (dysentery amoeba) Size: 12–35 mm (usually the size of 3–4 erythrocytes). but never erythrocytes.13) The invasive form measures 20–35 mm. Shape: oval or elongated. 4. histolytica can be found in liquid or diarrhoeal faeces: an invasive form and a non-invasive form. Invasive form (see Fig. 4. Inclusion bodies: numerous and varied (bacteria. Motility: moves in one direction. rather irregular. Non-invasive form (see Fig.14 Non-invasive form of Entamoeba histolytica trophozoite Identification of motile forms of amoebae Entamoeba histolytica (Figs 4. 4. Fig. Parasitology 113 Fig. often non-motile or moving very slowly.4. Cytoplasm: both the ectoplasm and the endoplasm are granular and difficult to differentiate. Entamoeba coli (Fig. putting out blunt pseudopodia in all directions. which may contain vacuoles. when not moving. cell debris).14) The non-invasive form measures 12–20 mm.15 Entamoeba coli trophozoite . It is non-pathogenic.13 Invasive form of Entamoeba histolytica trophozoite Fig. Two motile forms of E. It thrives in the intestinal cavity where it eats bacteria or other material that can be seen inside the vacuoles. round. elongated and changing. It has vacuoles containing more or less digested erythrocytes (1–20 of different sizes) indicating haematophagous (bloodeating) activity and so pathogenic capability. 4.13 and 4. Cytoplasm: the ectoplasm is transparent. 4. a pseudopodium pushes forward and the endoplasm flows quite rapidly into it.15) Size: 20–40 mm (usually bigger than E. Shape: when moving. histolytica).

yeast cells and varied debris. 4. visible after staining with iodine solution. Fig. Nucleus: a large oval karyosome next to a group of granules.16 Entamoeba hartmanni trophozoite Endolimax nanus (Fig. Inclusion bodies: various (mainly bacteria). histolytica but never contains erythrocytes. dense.19) Size: 6–15 mm. Shape: round. Motility: many small rounded pseudopodia moving slowly in all directions. butschlii amoebae are rarely seen in stools. Fig. Iodamoeba butschlii (Fig. rounded or finger-shaped pseudopodia.16) Size: always less than 10 mm (about the size of an erythrocyte). 4.4 Features for the differential diagnosis of Entamoeba histolytica and E. histolytica In a definite direction Fairly motile Transparent.18 Iodamoeba butschlii trophozoite Size: 10–15 mm. quite different from endoplasm Erythrocytes if haematophagous Invisible Regular membrane Small. histolytica. 4. If necessary. I. no erythrocytes Visible (nuclear membrane is like a bead necklace) Irregular membrane Large. Inclusion bodies: bacteria. If a trophozoite moves quickly in one direction and projects pseudopodia rapidly. Other species of amoeba do not usually move like this.17 Endolimax nanus trophozoite Fig.4 summarizes the features used for the differential diagnosis of Entamoeba histolytica and E. buffered methylene blue can be used to stain the nucleus for confirmation. Table 4.18) Fig. clear. coli. the karyosome large and eccentric. 4. leaf-like. large vacuoles. visible after staining with iodine solution. If the trophozoite moves as described and if erythrocytes are present in the cytoplasm. 4. 4. it can be assumed that it is E. Motility: very slow.19 Dientamoeba fragilis trophozoite Dientamoeba fragilis (Fig. central Haphazard Non-motile or barely motile Little or no differentiation from endoplasm Bacteria. . Nucleus: karyosome similar to an ink-spot. without staining. There may be distinct vacuoles. The membrane is irregular and granular (like a bead necklace).114 Manual of basic techniques for a health laboratory Table 4. coli Nucleus: visible in the fresh state.17) Size: 6–10 mm. Entamoeba hartmanni (Fig. Cytoplasm: very granular with small vacuoles. it is probably Entamoeba histolytica. 4. All characteristics similar to those of E. coli Feature Motion Motility Ectoplasm Inclusion bodies Nucleus (fresh state) Nuclear membrane (after staining with iodine solution) Karyosome E. eccentric E. 4. Shape: compact.

difficult to see. in a spiral. Motility: moves in one definite direction. 4. 4. can appear in an active flagellate vegetative form or as inactive cysts. Nuclei: two large oval nuclei. q q Trichomonas hominis (Fig. Parasitology 115 Motility: either non-motile (most often) or very motile (in very fresh fluid stools). Flakes of mucus in fluid stools often contain clusters of large numbers of G. Cytoplasm: clear ectoplasm. Chilomastix mesnili (Fig. visible after staining with iodine solution. Important: q The characteristic movement is seen only in fresh liquid stools.4. Flagella: usually four. sometimes turning in a loop (fluid stools). seeming to vibrate. faintly visible. Shape: triangular and tapered at one end. appears twisted. intestinalis. T. or is hardly motile. hominis is the most resistant flagellate. Shape: oval with two pointed poles. intestinalis). Identification of motile forms of flagellates All of these parasites. Motility: either moves forward in small rapid jerks in a definite direction. Undulating membrane: present on one side only. with pseudopodia similar to the blades of an electric fan. Motility: whirls and turns in all directions. Nucleus: one nucleus. karyosomes split into 4–6 granules (membrane hardly visible).21) Size: 10–15 mm (slightly smaller than G. with the exception of Trichomonas hominis. Shape: rather elongated front view: similar to a pear side view: spoon-shaped.20 Giardia intestinalis trophozoite . Giardia intestinalis (Fig. extremely motile (a rapid wavy movement). Fig. Nucleus: one or two nuclei. 4. The vegetative and cystic forms of G. 4. quickly becomes nonmotile under the coverslip.22) Size: 10–15 mm.20) Size: 10–18 mm (size of two erythrocytes). intestinalis are often found together in soft stools. Inclusion bodies: bacteria. It remains motile even in old stools.

23) Size: about 50 mm. a sort of mouth that contracts and expands. drawing in cell debris. 4.22 Chilomastix mesnili trophozoites Cytoplasm: greyish-green with: — towards the tapered end: a distinct spiral marking. without a lid. Nuclei: a large kidney-shaped nucleus next to a small round nucleus. Nucleus: one nucleus. Important: If stools are left exposed to the air. crossing the field in a definite direction and sometimes turning in circles.116 Manual of basic techniques for a health laboratory Fig. organisms of the infusoria type may fall on to them from the atmosphere. easily visible in unstained preparations. These may be confused with Balantidium coli. which move with rapid strokes. Cytoplasm: transparent. with one pole more rounded than the other. Identification of motile forms of ciliates Balantidium coli (rare) (Fig. “Mouth”: the cytostome.21 Trichomonas hominis trophozoites Fig. around which the flagellate turns (figure-of-eight) — near the rounded end: a mouth-like cleft (faintly visible cytostome). Shape: oval. 4. 4. . Cilia: covered with many small cilia. Motility: moves very rapidly in stools.

23 Balantidium coli trophozoite Leave freshly prepared stains for 3 days before use. 53) Methanol. 0. fix it by covering the slide with methanol for 3 minutes. Examine in more detail with the ¥40 objective. Put a coverslip on the slide and place it on the microscope stage. Method 1. Note: q q q Fig. 2. Avoid carrying over one staining solution to another. 4. 2. Prepare a thin faecal smear in sodium chloride solution on a clean slide. 1% solution (reagent no. Pipette 1 ml of diluted Field stain B onto the slide. intestinalis also stain blue and their nuclei stain red. 4. Emulsify a small portion of stool in 1% eosin solution on a clean slide. The cytoplasm and flagella of trophozoites of Giardia intestinalis stain blue and their nuclei stain red. Use the ¥10 objective to examine the smear systematically for unstained trophozoites and cysts. q q Method 1. followed by 1 ml of undiluted Field stain A. 6. 3. Mix well by tilting the slide and allow to stain for 1 minute. 3. Tip off the methanol. Wash the slide in water and allow it to air-dry. Scan the smear. 25): — Field stain A (undiluted) — Field stain B (diluted one part of stain in four parts of distilled water) Sodium chloride. Spread over an area of approximately 2 cm ¥ 1 cm.4. . 7. Parasitology 117 Rapid Field stain for faecal trophozoites Materials and reagents q q q q Microscope Microscope slides Slide rack Field stain (reagent no. 23). Once the smear is dry. Use rainwater to prepare the stains if the local well-water supply is too salty. 5. Examine the slide using the ¥100 oil-immersion objective. Cover the jars containing the staining solutions to prevent evaporation and absorption of dust. q Eosin stain for faecal trophozoites and cysts Materials and reagents q q q q q Microscope Microscope slides Slide rack Coverslips Eosin.85% solution (reagent no. particularly around the edges. Cysts of G.

sometimes irregular. Chromatoid bodies: oblong.3. not found in all cysts. The main thing is to differentiate between them and the cysts of E. Importance of cysts The clinical importance of cysts varies from country to country. Shape: round. Nuclei: 1–8 nuclei: membrane — irregular. Measurement of cysts is useful for the correct identification of species. often eccentric. 4. thick in parts. Note: If 1% eosin solution is not available. They are small. Cytoplasm: pale yellow after staining with iodine solution. Healthy persons may be asymptomatic carriers of cysts and are. flagellates and ciliates.118 Manual of basic techniques for a health laboratory The eosin stain provides a pink background against which unstained trophozoites and cysts are clearly visible. one at either pole. histolytica. Cytoplasm: yellowish-grey after staining with iodine solution. The cyst is the infective form of the organism. Identification of cysts of amoebae Entamoeba histolytica (Fig. 4. slightly larger than the cyst of E. 4. 4. therefore. Nuclei: 1–4 nuclei: membrane — thin. diffuse. histolytica). . regular. “dirty” appearance. E. Vacuole: sometimes a very large vacuole (stained reddish-brown by iodine solution) compressing two nuclei. not found in all cysts. Some of the features used in the identification of these cysts and those of other intestinal protozoa are illustrated in Fig. bright (as compared with E. Vacuole: sometimes a large glycogen vacuole (stained reddish-brown by iodine solution) in young cysts with one or two nuclei. not a perfect circle karyosome — large. rounded at ends (sausage-shaped). histolytica). Shape: round or slightly oval.2 Identification of cysts Cysts are the resistant forms of certain intestinal amoebae. Entamoeba coli (Fig. circular karyosome — small. central (like a black dot). a public health hazard. use a drop of Field stain B (see above). compact. Chromatoid bodies: sharp or jagged ends (dagger-shaped or needle-shaped).25) Size: 12–15 mm (1–2 erythrocytes). Giardia intestinalis and Balantidium coli.24. round and non-motile and may have one or several nuclei.26) Size: 12–20 mm (1–2 erythrocytes. histolytica may cause dysentery. The most important problem in the laboratory is the precise identification of cysts of Entamoeba histolytica. Identification of cysts of other amoebae that do not cause disease may be difficult. granular.

4. Parasitology 119 Fig.24 Features for the identification of cysts of intestinal protozoa . 4.

4. Iodamoeba butschlii (Fig.27 Entamoeba hartmanni cysts Size: 8–10 mm.28 Endolimax nanus cysts Shape: varies (round. Nuclei: 1–4 nuclei: membrane — cannot be seen karyosome — large. histolytica (see above). Vacuole: a very large glycogen vacuole (stained reddish-brown by iodine solution. 4. Shape: more or less oval. 4. identical to those of E. Endolimax nanus (Fig. 4. Cytoplasm: clear. pressed against a cluster of granules. stained yellow by iodine solution. . often taking up half of the cyst. without granules. irregular outline. Fig. 4. Nucleus: almost always a single nucleus: membrane — cannot be seen karyosome — very large.25 Entamoeba histolytica cysts Fig. hence the name Iodamoeba).28) Fig.29) Size: 8–10 mm. oval or irregular). 4.27) Size: 4–8 mm (same diameter as an erythrocyte). Nuclei: 1–4 nuclei.26 Entamoeba coli cysts Entamoeba hartmanni (Fig. Dientamoeba fragilis Not found in cyst form. oval.120 Manual of basic techniques for a health laboratory Fig. 4.

31 Chilomastix mesnili cysts Fig. folded in two or S-shaped.30) Size: 8–12 mm. 4. Cytoplasm: granular. 4. the second wall is the membrane of the cytoplasm. 4. greenish.29 Iodamoeba butschlii cysts Fig. hair-like line. one pole tapered (similar to a pear). Fibril: shiny. placed lengthwise in the centre of the cyst (adjust the microscope). Shell: thin. 4. Identification of cysts of ciliates Balantidium coli (Fig. Often the trophozoite form (see page 119) can be seen faintly inside. large nucleus: membrane — clearly seen.30 Giardia intestinalis cysts Fig. shiny when unstained. Shell: often appears to be thick with a double wall. 4. Fig. Parasitology 121 Identification of cysts of flagellates Giardia intestinalis (Fig. Nuclei: a large kidney-shaped nucleus next to a small round nucleus. pale yellowish-green or bluish after staining with iodine solution. 4. Shape: oval. central.32) Size: 50–70 mm (the size of an Ascaris lumbricoides egg).31) Size: 6–8 mm. thick in parts karyosome — small and central. Shape: round.32 Balantidium coli cyst . Nuclei: 2–4 oval nuclei (not clearly seen): membrane — very fine karyosome — small.4. Shape: round. one pole more rounded than the other. 4. Cytoplasm: clear. Nucleus: a single. double wall. faintly coloured. Fibril: twisted. Chilomastix mesnili (Fig. filled with inclusion bodies. like a curled hair.

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Fig. 4.33 Types of coccidia a: Containing four sporozoites and occasionally a few large granules clustered at one pole; b: containing one large round granular cell; c: containing refractive granules completely filling the interior.

Coccidia (Fig. 4.33) Coccidia are protozoa that may be parasites of humans (without causing any significant pathogenic effects) or may be found in transit in the stools of people who have consumed infected food (fish, rabbit, etc.). They appear in the stools in a form resembling cysts (and are called oocysts or sporocysts). Size: 15–20 mm, depending on the species. Shape: an elongated oval, sometimes tapered at one pole. Colour: colourless and transparent (or occasionally pale yellow). Shell: a quite distinct, slightly shiny double line; sometimes an operculum is present at one pole. There are three types of coccidia (see Fig. 4.33): (a) containing four sporozoites (small banana-shaped rods), each with a small round nucleus; sometimes a few large granules are clustered at one pole; (b) containing one large round granular cell; (c) containing shiny granules completely filling the interior. Microscopic examination of cysts Preparation in saline wet mount Cysts can be seen as transparent shiny globules standing out clearly against a grey background. They have well-defined shells. Using the ¥ 40 objective, look for shiny round objects with a diameter roughly equal to 1–3 erythrocytes. Chromatoid bodies Look also for chromatoid bodies (rod-shaped structures). Chromatoid bodies are more distinct in saline mounts than in iodine mounts. These bodies are characteristic in appearance and occur in cysts of Entamoeba histolytica and E. coli. The rodshaped chromatoid bodies of E. histolytica have blunt rounded ends; those of E. coli have pointed ends. These chromatoid bodies are seen less frequently in cysts of E. coli than in those of E. histolytica. Nuclei Nuclei are not easily visible in saline mounts but are clearly seen in iodine mounts. The appearance of the nucleus is important in differentiating between species of amoeba. Therefore, if cysts (or cyst-like bodies) are seen in the saline mount, examine an iodine mount.

4. Parasitology

123

Measurement Accurate measurement of cysts is essential for their correct identification. Measure any cysts you find; if possible use a calibrated graticule in the eyepiece (see section 3.1.1, page 56). Preparation in iodine wet mount Iodine mounts are used to detect cysts of amoebae and flagellates. Cysts can be detected with the ¥ 10 objective. Use the ¥ 40 objective to see the characteristics of the cysts and measure them to ensure correct identification. Iodine stains the cytoplasm of the cysts yellow or light brown; nuclei are stained dark brown. When cysts of Entamoeba spp. are stained with iodine, the arrangement of the peripheral chromatin and the position of the karyosome can be seen. (If the peripheral chromatin is absent, the cyst is not a species of Entamoeba.) These peripheral chromatoid bodies stain light yellow and may not be very clear. Sometimes, young cysts contain glycogen; this stains dark brown with iodine. Staining flagellate cysts with iodine enables the fibrils (filaments) to be seen. Cysts of several different species may be found in the same stool specimen. Concentration If necessary, use the formaldehyde–ether sedimentation technique (see section 4.5.2) to examine a larger number of cysts for more certain identification. Eosin stain for faecal trophozoites and cysts See section 4.3.1, page 117. Modified Ziehl–Neelsen technique for staining oocysts of Cryptosporidium spp. Infections with Cryptosporidium spp. cause fever, abdominal cramps, diarrhoea and weight loss with an associated eosinophilia. In severe cases, a malabsorption syndrome may develop. Cryptosporidiosis causes self-limiting diarrhoea in children. It is a recognized cause of chronic diarrhoea in adults with lowered immunity, e.g. patients with acquired immunodeficiency syndrome (AIDS). Cryptosporidiosis should be suspected in patients with chronic diarrhoea and weight loss, for which no other cause can be found. Materials and reagents Microscope Microscope slides Slide rack Petri disk Cotton wool Sodium chloride, 0.85% solution (reagent no. 53) Formaldehyde, 37% solution (formalin) Carbol fuchsin for Ziehl–Neelsen stain (reagent no. 16) Acid–ethanol for Ziehl–Neelsen stain (reagent no. 5) Malachite green, 1% solution (see reagent no. 31) Methanol.

q q q q q q q q q q q

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Fig. 4.34 Cryptosporidium spp. oocysts

Method 1. Emulsify a small amount of faeces in saline on a clean slide. Spread over an area of approximately 2 cm ¥ 1 cm. 2. Allow the smear to dry before fixing in absolute methanol for 5 minutes. If the patient is known to be, or suspected of being, positive for human immunodeficiency virus (HIV), fix the smear in formalin vapour for 15 minutes by placing the slide in a Petri dish with a cotton wool ball soaked in formalin. 3. Flood the slide with carbol fuchsin for 5 minutes. Wash the stain off with water. 4. Flood the slide with acid–ethanol solution to decolorize until faint pink. Wash the slide in water. 5. Counterstain the slide with malachite green solution for 2 minutes. Wash in water and place in a slide rack to drain and dry. Examine the slide under the microscope using the ¥ 40 objective. Oocysts of Cryptosporidium spp. stained by this method may show a variety of stain reactions from pale pink to deep red. The oocysts measure 4–6 mm. The sporozoites within the oocysts have an outer rim of deep stained material with a pale centre (Fig. 4.34). This differentiates oocysts from some yeasts which may stain red but have a homogeneous smooth appearance. Note: Cryptosporidium spp. belong to a group of parasites called coccidia (see page 122). Other parasites of this group are: — Isospora belli — Toxoplasma gondii — Plasmodium spp. The oocysts of Cryptosporidium spp. are highly resistant to disinfecting agents. Features not to be mistaken for cysts Fungi (Fig. 4.35) Size: 5–8 mm. Shape: oval, often with buds. Colour: reddish-brown after staining with iodine solution. Content: often an eccentric cluster of 3–6 small granules. Some forms of fungi (arthrospores) are rectangular, with a very clear oval cytoplasm inside. Blastocystis hominis (yeast) (Fig. 4.36) Size: 5–20 mm (average 10 mm).

4. Parasitology

125

Shape: round or oval, sometimes with angular irregular edges. Colour: very shiny when unstained; the vacuole is not stained by iodine solution, but the periphery is pale yellow. Content: one large vacuole taking up almost the whole cell; the compressed cytoplasm forms a granular ring round it. Some physicians request that the presence of B. hominis be reported, particularly in children’s stools.
Fig. 4.35 Fungi

Leukocytes (white blood cells) (Fig. 4.37) Size: 10–20 mm. Shape: round or slightly elongated, with an irregular outline. Nucleus: indistinct, sometimes with a star-shaped “false karyosome”. Content: shiny cytoplasm, clear and granular with tiny vacuoles. Pus (Fig. 4.38) Pus appears to the naked eye as opaque, greyish streaks (not transparent like mucus). Under the microscope it appears as a mass of degenerated leukocytes. The presence of pus should be reported as it is a sign of infection.
Fig. 4.36 Blastocystis hominis

Fig. 4.37 Leukocytes

Fig. 4.38 Pus

4.4 Intestinal helminths
Helminth infections cause a variety of clinical symptoms including abdominal cramps, fever, weight loss, vomiting, appendicitis, blood loss, anaemia and eosinophilia. There are three groups of medically important helminths: — nematodes (roundworms) — cestodes (tapeworms) — trematodes (flukes). Helminth infections are usually diagnosed by detecting eggs and larvae. Less frequently, infections are diagnosed by detecting adult worms (e.g. Ascaris lumbricoides and Enterobius vermicularis) or proglottids (segments) of adult worms (e.g. Taenia saginata and T. solium). However, for most helminth infections, eggs are used for identification.

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4.4.1 Identification of eggs
The characteristics used to identify eggs of helminth species are as follows: Size The length and width are measured and are generally within a specific range. Shape Each species has its own particular shape. Stage of development when passed The eggs of some species consist of a single cell, some eggs have several cells, and some eggs are usually embryonated, i.e. they contain a larva. Occasionally, if stool specimens are 1–2 days old, eggs may develop to more advanced stages. Ascaris lumbricoides (roundworm) eggs usually have only one cell when passed in the faeces; however, the single cell may divide and, in specimens over 12 hours old, eggs with two or four cells may be seen. Ancylostoma duodenale or Necator americanus (hookworm) eggs present in specimens that are several hours old may contain 16, 32 or more cells. After 12–24 hours, the eggs may be embryonated and later still the larvae may hatch. When observing the stage of development of helminth eggs, be sure that the stool specimen is freshly passed. If it is several hours or a day old, expect to see changes in the stage of development of some species. Ideally only fresh samples should be accepted for diagnosis. Thickness of the egg shell The eggs of some species such as Ascaris lumbricoides have thick shells, whereas others such as Ancylostoma duodenale or Necator americanus have thin shells. Colour The eggs of some species such as Ancylostoma duodenale, Necator americanus and Enterobius vermicularis are colourless, whereas others such as Ascaris lumbricoides and Trichuris trichiura are yellow or brown. Other characteristics The presence of characteristics such as opercula (lids), spines, plugs, hooklets or mammilated outer coats can also be aids to identification. If an egg or an object that looks like an egg is found, the above-mentioned characteristics should be carefully observed in order to make a specific identification. Occasionally, atypical or distorted eggs are seen. In such cases, it is necessary to look for more typical forms in order to make a reliable diagnosis. Remember that more than one species of helminth may be present in a patient. Measurement of eggs
q

1 micrometre (1 mm) = 0.001 mm. The size in mm given in this manual is that of the long side of the egg. The size can be estimated by comparison with that of an erythrocyte, which measures 7.5–8 mm.

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127

q

The size can be assessed in relation to the microscope field: — if a ¥ 10 objective is used, the egg takes up about one-tenth of the field — if a ¥ 40 objective is used, the egg takes up about one-third of the field.

q

The egg can be measured by inserting a micrometer scale slide in the eyepiece of the microscope. One division of the scale using the ¥ 10 objective and the ¥ 10 eyepiece = 1 mm. Another method of measuring is to compare the egg with one of another species common in the locality whose size under the microscope is known (e.g. Ascaris lumbricoides).

q

How to recognize eggs The method recommended is:
q q

Establish the probable identity of the egg from its general appearance. Make a systematic study of all the characteristics of the egg to confirm its identity. In order to gain experience (if possible, under the guidance of an instructor): — study the different eggs found in your locality; — identify, one by one, all the characteristics of each egg as described in this manual.

Table 4.5 lists the helminth species whose eggs are found in stools. The terms used for the identification of helminth eggs and a key to their identification are given in Figs. 4.39 and 4.40, respectively. Fig. 4.41 shows the relative sizes of helminth eggs. Ancylostoma duodenale Size: 50–80 mm. Shape: oval with rounded slightly flattened poles (one pole often more flattened than the other). Shell: very thin; appears as a black line. Content: varies according to the degree of maturity. Colour: pale grey; dark brown after staining with iodine solution. Type A (in fresh stools) (Fig. 4.42) Four, eight or 16 grey granular cells, clear but not shiny (blastomeres). Type B (in stools a few hours old) (Fig. 4.43) A uniform mass of many small grey granular cells. Type C (in stools 12–48 hours old) (Fig. 4.44) The whole of the egg is filled by a small larva (the future worm), wrapped around itself. The egg is “embryonate”. Ascaris lumbricoides There are four types of Ascaris egg:
q q

A: fertilized egg with double shell. B: unfertilized egg with double shell.

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Table 4.5 Helminth species whose eggs are found in stools
Scientific name Ancylostoma duodenale Ascaris lumbricoides Clonorchis sinensis Dicrocoelium dendriticum, Dicrocoelium hospes Diphyllobothrium latum Dipylidium caninum Enterobius vermicularis Fasciola gigantica Fasciola hepatica Fasciolopsis buski Heterophyes heterophyes Hymenolepis diminuta Hymenolepis nana Metagonimus yokogawai Necator americanus Opisthorchis felineus Paragonimus westermania Schistosoma haematobium Schistosoma intercalatum Schistosoma japonicum Schistosoma mansoni Schistosoma mekongi Strongyloides stercoralis c Taenia saginata Taenia solium Trichostrongylus (various species) Trichuris trichiura
a b c

Geographical distribution Worldwide Worldwide South-east Asia Worldwide Worldwide Worldwide Worldwide Worldwide Worldwide Eastern and southern Asia South-east Asia, eastern Mediterranean Worldwide Worldwide Eastern and southern Asia, central and eastern Europe Worldwide Eastern and southern Asia, central and eastern Europe Central Africa, South America, eastern and southern Asia
b

Africa, eastern Mediterranean Africa Eastern and southern Asia Africa (south of the Sahara), Central and South America, the Caribbean South-east Asia Worldwide Worldwide Worldwide Asia Worldwide

Found mainly in sputum. Found mainly in urine. Found mainly as larvae in stools.

q q

C: semi-decorticated fertilized egg (less frequent). D: semi-decorticated unfertilized egg (very rare).

Type A. Fertilized egg with double shell (Fig. 4.45) Size: 45–70 mm. Shape: oval or sometimes round. Shell: the two shells are distinct: — the external shell is rough, brown and covered with small lumps (mamillated) — the internal shell is smooth, thick and colourless. Content: a single round granular central mass. Colour: external shell — brown; content — colourless or pale yellow.

39 Terms used for the identification of helminth eggs .4. 4. Parasitology 129 Fig.

not embryonated Flattened operculum? Yes Paragonimus westermani (65–120 µm x 40–50 µm) Echinostoma sp. (80–120 µm x 55–90 µm) Fasciola hepatica (130–145 µm x 70–90 µm) Fasciolopsis buski (125–140 µm x 70–90 µm) No Length >120 µm Ovoid. golden yello w. yellowish brown With plugs Clearly protruding plugs (50–65 µm x 20–30 µm) No clearly protruding plugs.130 Manual of basic techniques for a health laboratory Eggs Not encapsulated With or without plugs? Encapsulated? Encapsulated (30–40 µm x 25–50 µm) Dipylidium caninum Without plugs With or without operculum? With operculum Without operculum With or without spine? Length of egg? Length <35 µm With or without shouldering? Without shouldering Elongated (25–35 µm x 8–12 µm) Thin shell (25–30 µm x 15–20 µm) Opisthorchis felineus (posterior end with pointed terminal knob) Metagonimus yokogawai Clonorchis sinensis (posterior end broad and round) Heterophyes heterophyes (small knob at posterior end) Diphyllobothrium latum With shouldering Marked shouldering (25–45 µm x 10–22 µm) Less distinct shouldering. thick shell.40 Key to the identification of helminth eggs Capillaria philippinensis . thick shell. not embryonated (55–80 µm x 40–50 µm) Ovoid. golden brown. lemon-shaped (35–45 µm x 20–30 µm) Slightly narrowing in middle? No Trichuris trichiura Capillaria hepatica Yes Fig. 4. thick shell. broad operculum (25–30 µm x 15–17 µm) Length 35–120 µm Ovoid.

4. Parasitology 131 .

41 Relative sizes of helminth eggsa a Schistosoma intercalatum and S. . mekongi have been omitted.132 Manual of basic techniques for a health laboratory Fig. 4.

roundish. 4.47 Semi-decorticated fertilized Ascaris lumbricoides egg Fig.48 Semi-decorticated unfertilized Ascaris lumbricoides egg Type B. 4. or Fasciolopsis buski eggs. 4. 4. Shell: the two shells are indistinct: — the external shell is brown and puffy. 4. thin and colourless (double line).45 Fertilized Ascaris lumbricoides egg with double shell Fig. colourless. . 4. very shiny granules. Content: the egg is full of large. 4. Parasitology 133 Fig. Type D.47) Similar to type A but without the external shell.46) Size: 45–90 mm (larger than type A). thick and colourless (or very pale yellow). Content: large. Fasciola spp. colourless. round. smooth. Caution: Do not confuse type D with Ancylostoma duodenale. Unfertilized egg with double shell (Fig.48) Shell: single. Type C. with rather jagged lumps — the internal shell is thin (one or two lines may be visible). smooth. Semi-decorticated fertilized egg (Fig.4. Semi-decorticated unfertilized egg (Fig. Content: a single round.46 Unfertilized Ascaris lumbricoides egg with double shell Fig. Shape: more elongated than type A (elliptical or irregular).44 Ancylostoma duodenale egg in stools 12–48 hours old Fig. 4. 4. 4. granular central mass.43 Ancylostoma duodenale egg in stools a few hours old Fig.42 Ancylostoma duodenale eggs in fresh stools Fig. shiny granules. Shell: single.

Content: a well-organized ciliated embryo. 4. Content: a ciliated embryo. Fig. Type A. 4. Shape: distinctive. Operculum: easily visible at the narrow end of the egg. The eggs of the flukes are not digested and although they appear in the stools.134 Manual of basic techniques for a health laboratory Clonorchis sinensis (Fig. at the opposite end to the operculum. Content: an indistinct dark yellow oval mass. Operculum: easily visible. often with 1–4 shiny globules. fitting into a thickened rim of the shell. egg in passage O: operculum. Size: 35–50 mm.50 Dicrocoelium sp. 4.50) Shell: yellow. Content: a mass of small cells around a large central cell. Repeat the examination 8 days later. Colour: shell — yellowish-brown. Fig. Boss: very small. 4. Eggs from infected patient (very rare.51) Shell: uniform dark brown. Colour: pale yellow. Fig. Dicrocoelium spp. the patient is not infected. Tell the patient not to eat liver or liver products in the meantime. Eggs in passage1 (form most often found. 4. 4. Fig. orange or light brown. Boss: a small knob at the wide end of the egg.52) Size: 55–80 mm. Diphyllobothrium latum (Fig.49 Clonorchis sinensis eggs B: boss.52 Diphyllobothrium latum egg B: boss. Shell: thick.49) Size: 25–45 mm. 4. rather asymmetrical. 1 Observed when the patient has eaten sheep or beef liver infected by the flukes. content — pale yellow. smooth and yellow. Fig. 4. orange or light brown.51 Dicrocoelium sp. O: operculum. egg from infected patient Type B. Shape: oval. Shell: fine and smooth but quite thick (double line). Shell: smooth and thick. . Fig. Shape: oval. Operculum: scarcely visible when not raised.

a small curled-up larva. vermicularis eggs are usually more easily found in the folds of skin around the anus than in the faeces (see below). Place the spoon handle against the underside of the slide (Fig. 0. Atlanta. Education and Welfare.53) Dipylidium caninum eggs are found in clusters of 6–20 enclosed in a fine membrane. 53).54 Enterobius vermicularis eggs Fig. They rarely appear in the stools. Enterobius vermicularis (Fig. Materials and reagents Microscope Microscope slides Test-tubes Pasteur pipette Adhesive cellophane tape Spoon 10 cm long or.56).85% solution (reagent no. United States Department of Health. Colour: colourless. 4. Fig. granular mass in the shape of an irregular oval. Colour: yellow or pale grey. Size: 30–40 mm (the cluster is 150–300 mm). sticky side down. Content: a single uniform granular mass with three pairs of shiny hooklets arranged in the shape of a fan. 4. Parasitology 135 Dipylidium caninum (Fig. 4. Fig. Place a strip of cellophane tape. E.4. as shown in Fig. without striations. 4. Shape: oval but clearly asymmetrical (flattened on one side.55. 4.53 Dipylidium caninum eggs q q q q q q q q Method1 1. Shape: round. Centers for Disease Control. rounded on the other). on a slide. 4. Laboratory procedures for the diagnosis of intestinal parasites. or (b) the embryo of the worm.54) Size: 50–60 mm. GA. 1969:138. 2. Content: either (a) a small. . a wooden tongue depressor Cotton wool Sodium chloride. but a double line is visible. 4. better. Technique for the collection and examination of eggs Principle The eggs of Enterobius vermicularis (pinworm) are usually collected (particularly in children) from the folds of skin around the anus. Brooke M. Shell: thick and slightly granulated. Shell: smooth and thin. 4.56 Positioning the spoon handle underneath the slide 1 Based on Melvin D.55 Preparation of slide for collection of pinworm eggs Fig.

Press the end of the spoon covered with tape against the skin round the anus in several places (Fig. 7.57. using the ¥ 10 objective. 6. Examine under the microscope with the condenser aperture reduced. 4. Separate the patient’s buttocks with your left hand. Gently pull the tape away from the slide and loop it over the end of the spoon handle. 4.59).57 Looping the tape over the end of the spoon handle Fig. Hold the completed tape swab in your right hand. sticky side down (Fig. Look for eggs of E. vermicularis (see Fig. Take the slide and fold the tape back on to it. 4. Fig.58). Make sure that the tape is firmly stuck flat to the slide by pressing it with a piece of cotton wool (Fig. 4. 4. 4. pressing the slide firmly against the spoon.136 Manual of basic techniques for a health laboratory 3. 4.58 Technique for collecting pinworm eggs from an infant .60). 8.54). as shown in Fig. 4. 5.

Transfer it to a slide (Fig. 4.60 Making sure that the tape is firmly stuck to the slide Fig.62). Rinse the swab well in the solution (Fig.61 Alternative technique for collecting pinworm eggs from an infant Fig.63). 4.5 ml (10 drops) of sodium chloride solution. Dip the swab into a test-tube containing about 0. 4. Draw up the liquid with a Pasteur pipette. If no cellophane tape is available. . use a cotton wool swab to wipe around (but not inside) the anus (Fig.62 Transferring the sample to a testtube Alternative method 1. 4. 4.59 Transferring the sample to the slide Fig. Parasitology 137 Fig. 3.61). 4. cover with a coverslip and examine under the microscope as described in step 8 above. 4. 2.4.

O: operculum. but usually present in greater numbers in the stools. 4. Thickening of a small part of the cell wall at the other pole. Operculum: slightly smaller than F. single line.63 Transferring the sample to a slide Fig.49). Only small numbers of eggs are found in the stools (a search can be made by duodenal aspiration in doubtful cases).65 Fasciolopsis buski egg T: thickening.66) Similar to the eggs of Clonorchis sinensis (see Fig.64). sinensis. Colour: ranges from yellow to dark brown. 4. hepatica. 4. sinensis. hepatica. . Heterophyes heterophyes (Fig. Fasciola hepatica (Fig. Shell: smooth and fine with a double line. granular nuclei (adjust the focus). the operculum does not overlap. Shape: oval. Shell: thinner than F. Content: a mass of large indistinct cells with clear. Size: 25–30 mm. Boss: tiny and wart-shaped.64 Fasciola hepatica egg T: thickening. Shape: more oval than C. O: operculum. Size: 125–140 mm.138 Manual of basic techniques for a health laboratory Fig. 4. 4. Shape: oval with rounded poles. with a marked thickening of the wall at the opposite pole to the operculum. not always visible. Content: cells may be shiny with one clear cell in the centre of the egg. 4. at the wider end of the egg.64) Size: 130–145 mm. Other features: finely marked operculum at one pole. the cell wall may be visibly retracted. Fasciolopsis buski (Fig. Shell: slightly thicker than that of C. 4.65) Very similar to the eggs of Fasciola hepatica (see Fig. 4. Fig.

Boss: tiny or invisible. Shell: external shell thin with transverse lines. Content: a ciliated embryo. Important: Record whether there are many or few eggs present.66). nana). Shell: thicker than C. Colour: transparent or pale yellow. with filaments coming away from both poles (reduce the intensity of the microscope light source to see them). Shape: oval. Colour: yellow to dark brown.68) Size: 40–60 mm. Hymenolepis diminuta (Fig. 4.49 and 4. Content: rounded mass (embryo) with six shiny hooklets arranged in fan shape and often some well-defined granules in the centre. sometimes with large shiny granules (unfertilized) or a ciliated embryo. Shell: double. at the narrower end of the egg. 4.69 Metagonimus yokogawai egg Fig. Operculum: more rounded than in H. 4. mixed with granules occupying the space between the two membranes. external membrane thin and internal membrane often thicker at the poles. 4. 4.4. Shape: oval. Size: 25–30 mm.67) Rare species (found in children’s stools). Hymenolepis nana (Fig. 4. 4. sinensis. Metagonimus yokogawai (Fig.68 Hymenolepis nana egg . Fig. almost round. Size: 70–90 mm (much larger than H.66 Heterophyes heterophyes eggs Fig. Content: a rounded embryo containing six hooklets arranged in fan shape. Colour: very pale grey. heterophyes.69) Similar to the eggs of Clonorchis sinensis and Heterophyes heterophyes (see Figs. heterophyes.67 Hymenolepis diminuta egg Content: a mass of cells. Parasitology 139 Fig. 4. Shape: round. internal shell very thick without filaments. with no marked shouldering. sinensis and H. overlapping less than in C.

Content: clear central space surrounded by squarish cells. Size: about 200 mm. yokogawai: thicker shell. see section 7. 4. . 4. felineus. sinensis. duodenale).74) Eggs found in urine (for detection. with one well-rounded pole.8) and occasionally in stools. S. some eggs are asymmetrical. Shape: spindle-shaped. Shape: oval with rounded flattened poles (more flattened than in A. Boss: rarely visible. Spine: terminal and situated at the other pole. 4. Schistosoma bovis (Fig. Colour: golden brown. Size: 60–80 mm (slightly longer than A. 4. Shape: slightly narrower at the base and with less shouldering than C. sinensis). H. Content: always contains at least eight cells (never four like A. duodenale in fresh stools). Heterophyes heterophyes and Metagonimus yokogawai: q q q q O.42). duodenale). It is very difficult to differentiate between the eggs of O.73) Eggs found in the stools of patients who have eaten infected beef. 4. felineus: narrow. often asymmetrical. 4.73 Schistosoma bovis egg Spine: long terminal spine. 4. 4.70) Almost identical to the eggs of Ancylostoma duodenale (see Fig.70 Necator americanus egg Opisthorchis felineus (Fig.2. bovis does not cause disease in humans.72 Paragonimus westermani egg Eggs mainly found in sputum (if swallowed they pass into the stools). C. 4. operculum with distinct overlap. Shape: oval. M. Size: 25–35 mm (identical to C. Paragonimus westermani (Fig.71 Opisthorchis felineus eggs Content: a ciliated embryo. often slightly flattened on one side. sinensis. 4. with narrowed extremities extending beyond the embryo. Shell: distinct thickening at the opposite end to the operculum. Shape: oval. Operculum: quite distinct.72) Fig. Content: small round embryo lying in the centre of the egg but not filling it. heterophyes: squat shape. Size: 65–120 mm (smaller than the eggs of Fasciolopsis buski ). C. Fig.71) Similar to the eggs of Clonorchis sinensis (see Fig.140 Manual of basic techniques for a health laboratory Necator americanus (Fig. 4. sinensis: squat shape. sinensis. Schistosoma haematobium (Fig. Size: 110–150 mm. with an obvious rim. darker colour.49). boss rarely visible. Operculum: less overlap than C. Fig. Fig.

may be hidden by small granules often found on the surface of the egg. and shipped great distances. adjust the focus of the microscope). Schistosoma japonicum (Fig. Shape: oval. Colour: pale yellow. haematobium (sides particularly flattened towards the rounded pole). very thin. with one well-rounded pole and one conical pole. Colour: grey or pale yellow. The slides can be prepared in the field. 4. Fig. 4. 60–105 mesh Template. near the rounded pole.74). very thin. Size: 140–180 mm (slightly larger than S.75) Similar in appearance to S. Spine: lateral. S: spine. Content: a ciliated embryo surrounded by a membrane with two depressions or indentations. but found in stools. stored in microscope-slide boxes. 4. Materials and reagents q Flat-sided applicator stick. mansoni and certain other intestinal helminth infections. one on each side near the middle. surrounded by a membrane (internal shell) as in all Schistosoma spp. Colour: transparent or pale yellow.76 Schistosoma japonicum egg G: granules. nylon or plastic. 4. less broad than S. .76) Size: 70–100 mm. longer and more tapered than S. The technique is not suitable for diagnosing strongyloidiasis or infections with Enterobius vermicularis or protozoa. Content: a broad ciliated embryo.77 Schistosoma mansoni egg S: spine. Spine: terminal spine. Content: a well-formed broad ciliated embryo surrounded by a membrane (internal shell). plastic or cardboard Microscope Microscope slides Cellophane. wooden q q q q q Fig. haematobium. 4. stainless steel. Shape: spindle-shaped. S: spine. Shell: smooth.77) Size: 110–180 mm. Schistosoma intercalatum (Fig. Cellophane faecal thick-smear technique for diagnosis of Schistosoma mansoni infection (Kato–Katz technique) The Kato–Katz technique has proved to be an efficient means of diagnosing S. in strips 25 mm ¥ 30 mm or 25 mm ¥ 35 mm Fig. lateral and very small. Spine: difficult to see. Fig. 4. Shape: oval.74 Schistosoma haematobium egg S: spine. haematobium (see Fig. large and triangular (if hidden underneath. Schistosoma mansoni (Fig. 40–50 mm thick. stainless steel. Screen. almost round. 4.4. haematobium). Content: a broad ciliated embryo. for examination at a central laboratory if required. 4.75 Schistosoma intercalatum egg D: depression. Parasitology 141 Shell: smooth.

79). Remove the template carefully so that all the faecal material is left on the slide and none is left sticking to the template.5 g) of faeces on to a piece of scrap paper (newspaper is ideal). scrape across the upper surface of the screen to sieve the faecal sample . Always wear gloves. newspaper) Glycerol–malachite green solution (reagent no.78 Using an applicator stick. 8. Press the screen on top of the faecal sample. 39). 4.80). 4. 4. 2.g. 4.78). 6. Soak the cellophane strips in the glycerol–malachite green (or methylene blue) solution for at least 24 hours before use. Invert the microscope slide and press the faecal sample against the cellophane on a smooth surface (a piece of tile or flat stone is ideal) to spread the sample evenly. Using the applicator stick. Transfer the sieved faecal material into the hole of the template and level with the applicator stick (Fig. wipe it off with a small piece of absorbent tissue. Cover the faecal sample on the slide with a glycerol-soaked cellophane strip (Fig. 1. 9. Transfer a small amount (approximately 0. 4. 7. scrape across the upper surface of the screen to sieve the faecal sample (Fig.142 Manual of basic techniques for a health laboratory q q q q q Flat-bottomed jar Forceps Toilet paper or absorbent tissue Scrap paper (e. 5. Method Important: Care must be taken to avoid contamination during collection of stool specimens. 31) or methylene blue solution (reagent no. If any glycerol is present on the upper surface of the cellophane. 3. Place the template on a clean microscope slide. Fig.

Gently slide the microscope slide sideways while holding the cellophane.81 Strongyloides stercoralis eggs Fig. Shell: very thick. Do not lift the slide straight up or it may separate from the cellophane. . 4. be found in liquid stools (and occasionally in the formed stools of carriers of certain strains). Wipe off any excess glycerol with a piece of absorbent tissue to ensure that the cellophane stays fixed. Content: a thick larva curved around itself one or more times and sometimes motile. Taenia saginata and T. 4. Strongyloides stercoralis (Fig. with transverse lines (reduce the illumination). Parasitology 143 Fig. S. Preparation of the slide is now complete. saginata can also be collected from the skin around the anus (see page 136). Fig. duodenale).42). They may be found in stools and eggs of T. Shell: very thin.82(a)) The “eggs”1 of these two tapeworms are practically identical.4. solium (Fig. They may.81) S. Content: a round granular mass enclosed by a fine membrane. Size: 30–80 mm. Shape: oval with slightly flattened poles. Colour: pale grey. smooth. stercoralis eggs are rarely seen in formed stools because they hatch before evacuation to produce larvae. 4. 4. however. stercoralis eggs are very similar to those of Ancylostoma duodenale (see Fig. embryonated eggs that have lost their outer sac. Size: 50–80 mm (slightly smaller than A. dark brown after staining with iodine solution. 4.79 Filling the template with the sieved faecal sample 10. 4. With practice you can obtain perfect preparations.80 Covering the faecal sample with a glycerolsoaked cellophane strip 1 The correct name for these “eggs” is “embryophores”. appears as a black line. with three pairs of shiny lancet-shaped hooklets (adjust the focus). Shape: round.

83 Trichostrongylus sp.84) Size: 50–65 mm.82(b)). content — light yellowish-grey. with two layers. Other features: a rounded. 4. Content: masses of starch packed closely. The egg quickly develops into an embryo. Shell: thick in places.85 Starch granules from plants . asymmetrical. transparent plug at each pole. Trichostrongylus spp. eggs Fig.82 Taenia spp. very irregular. 4. Colour: whitish or greyish-yellow. Shell: very thin and smooth (similar to A.84 Trichuris trichiura eggs Fig. Trichuris trichiura (Fig. duodenale). Shape: round or oval and elongated. Size: 75–115 mm (slightly larger than A. 4. eggs a: Normal egg. with cracks. Other features: sometimes the egg is enclosed in a floating transparent sac (Fig. duodenale).144 Manual of basic techniques for a health laboratory Colour: shell — dark yellowish-brown.83) Quite similar to eggs of Ancylostoma duodenale (see Fig. 4. Fig. Content: a uniform granular mass (sometimes divided in old stools). (Fig. Features not to be mistaken for eggs Starch granules from plants (Fig. 4.42). 4. Content: a mass of at least 20 small round granular cells (in fresh stools). content — yellow. Shell: fairly thick and smooth. Colour: shell — orange. 4. Shape: oval. with one rounded pole and one narrower pole. Colour: yellowish-brown.85) Size: 50–100 mm. Shape: barrel-shaped. 4. Important: Specify whether there are many or few eggs present. 4. violet after staining with iodine solution. b: egg enclosed in a floating transparent sac. Fig.

yams and cassava. tapered at the other.89) Size: variable (can be any size). very shiny (several layers in the case of oil). oval or irregular (like a section of a tree trunk).90) Size: very variable (50–300 mm). Colour: pale yellow. beans.86) Size: 100–200 mm. Shape: perfectly round. often curved. 4. Colour: yellow. False shell: a circular ring. Shape: rather rigid. 4. 4. wide and clean-cut at one end. Fig. Content: lines radiating from the centre and visible near the rim. 4. Content: none. Shape: round. Colour: brownish-yellow or colourless.88 and 4. Content: a narrow empty central canal between two transparent shiny layers. Digested meat fibres (Fig. 4.87) Size: 20–100 mm.89 Oil droplets .4. Parasitology 145 These granules are the residue of starchy foods such as potatoes. Shape: oval or rectangular with rounded corners. 4. 4.87 Soap Fig. Air bubbles and oil droplets (Figs. 4. Content: transparent with no granulations or lines (or residual lines where meat is not properly digested).86 Digested meat fibres Fig. Soaps (Fig. nothing in the centre.88 Air bubbles Fig. Plant hairs (Fig.

in clothing or bed linen. 4. with a very pointed tail (males are less common). 4.5 cm. 4. solium (pork tapeworm) Length: total worm.146 Manual of basic techniques for a health laboratory Pollen grains and fungus spores (Fig. Other features: distinctive saw-like or rounded projections.91 Pollen grains and fungus spores Enterobius vermicularis (pinworm or threadworm) (Fig. Colour: white. Common helminths Fig. and are motile. where they can be collected with a strip of adhesive cellophane (see section 4. but single mature segments (1–3 cm long) or fragments of the chain (variable in length) are usually presented for examination. with a straight tail. page 135). Shape: distinctive geometrical shapes. Taenia saginata (beef tapeworm) and T. .4. Colour: pinkish. They may also be found in the folds of skin around the anus. What to examine: — their length — their shape — whether they are flat or segmented — whether they are cylindrical (round) — their internal structure.93) Length: male — 0. or during a surgical operation. 4. 4.91) Size: very variable.92) Length: male — about 15 cm. 4. with a curved tail.2 Identification of adult helminths Adult helminths brought to the laboratory for identification may have been found in stools.90 Plant hairs Ascaris lumbricoides (roundworm) (Fig. etc. Fig. 3–10 m.1. female — 20–25 cm. female — 1 cm.4. especially in children’s stools. depending on the geographical area and the local diet. Pinworms or threadworms are found in large numbers.

separate pieces may dry out and roll up.94 Segments of adult Taenia spp. untangle it.) Fig. 4.96). 3.94). Place the whole worm in a Petri dish (or on a plate) filled with water. solium). making them look like roundworms. transfer the worm little by little into another dish (Fig.93 Adult Enterobius vermicularis (pinworm or threadworm) Colour: ivory white (T.92 Adult Ascaris lumbricoides (roundworm) Fig. Examine a single segment gently flattened between two slides (Fig. Moisten them with water to restore their shape. To examine the head (scolex): 1. saginata) or pale blue (T. Important: If there is a delay in examination. 2.4. examine it under the microscope with the ¥ 10 objective or with a magnifying glass. 4. If at the end of a very narrow section (the neck) you find a swelling the size of a small pinhead. Parasitology 147 Fig. Examination Materials and reagents q Microscope or magnifying glass q q q Microscope slides Petri dish Forceps. .95). 4. (The head is rarely found. starting with the thicker end. 4. q Hold the slide against the light to observe and count the uterine branches with the naked eye. 4. 4. Using forceps. q Method Examine a chain of segments to observe the arrangement of the lateral pores (Fig.

4. occasionally found in a patient’s organs during a surgical operation. . or red if it contains blood. Length: 1.96 Using forceps to transfer a tapeworm Figure 4. 4.93).5 cm.98) A roundworm (resembles a piece of thread) similar to E. Hymenolepis nana and Dipylidium caninum. Other helminths found in stools The helminths described below are rarely found in the stools. 4.0–1.148 Manual of basic techniques for a health laboratory Fig.95 Flattening a segment between two slides Fig. They are. Ancylostoma duodenale and Necator americanus (hookworm) (Fig. solium and two less common tapeworms. Examine the head (scolex) under the microscope with the ¥ 10 objective. however. 4. Colour: white. vermicularis (see Fig. saginata and T.97 shows how to differentiate between T. Flukes are seen in the liver and intestines and hydatid cysts are observed in the liver and lungs.

Parasitology 149 Fig. 4.97 Features for the identification of tapeworms .4.

Large fluke Length: 2–3 cm. Small fluke Length: 0. Colour: reddish-brown or dull white.5–1. Length: 3–5 cm.99 Adult Trichuris trichiura (whipworm) Trichuris trichiura (whipworm) (Fig. Colour: transparent. 4.100) A flatworm with two suckers. it looks like a leaf. 4.101) Fig. Flukes (Fig.150 Manual of basic techniques for a health laboratory Fig. . Width: fairly broad. 4. (blood flukes) (Fig.99) A small thin worm that lives in the wall of the caecum or occasionally the rectum. Width: narrow.100 Flukes A small thin flatworm.98 Adult Ancylostoma duodenale and Necator americanus (hookworm) Fig. 4. 4. greyish-red. 4. Schistosoma spp.0 cm. Colour: white.

Parasitology 151 Fig. 4. Hydatid disease occurs preferentially in areas where sheep are bred. Shape: round. Size: about 150 mm. with one pole slightly flattened. Each schistosome has two suckers near the head.102 Hydatid cysts Length: 0. which then develop into hydatid cysts in the liver or lungs (Fig. The worms are 3–6 mm long. 4. Colour: white. irregular or oval. which is slightly longer.102). such as East and North Africa. South America. 4. . the Arabian peninsula. Content: fine granules and a distinct ring of 10–30 hooklets. Humans and livestock may become infected by accidental ingestion of the eggs. Echinococcus granulosus (hydatid cyst) Echinococcus granulosus worms are found in dogs. The flat male is rolled around the thread-like female.5–1.101 Schistosomes Fig. Colour: colourless and transparent.4.5 cm. Australia and New Zealand.

5.1 Flotation technique using sodium chloride solution (Willis) This method is recommended for the detection of eggs of Ancylostoma duodenale and Necator americanus (best method). or protozoal cysts or trophozoites.. 4. and Trichuris trichiura. . anaemia.) 4.5 Techniques for concentrating parasites Concentration techniques are used when the number of helminthic ova or larvae. Four different concentration techniques are described in this book: — the flotation technique using sodium chloride solution (Willis) — the formaldehyde–ether sedimentation technique (Allen & Ridley) — the formaldehyde–detergent sedimentation technique — the sedimentation technique for larvae of Strongyloides stercoralis (Harada– Mori). Length: up to 20 m. is small. or protozoal cysts or trophozoites. Fig. Important: Always make a direct microscopic examination of stools before preparing a concentration. Principle The stool sample is mixed with a saturated solution of sodium chloride (increasing the specific gravity). Infection occurs through eating raw or inadequately cooked fish and can result in intestinal obstruction. 64) Petroleum jelly Wax. 4. (Motile forms of protozoa are not found in concentrated preparations. 4. It is not suitable for the detection of eggs of flukes and Schistosoma spp. Taenia spp. larvae of Strongyloides stercoralis.152 Manual of basic techniques for a health laboratory Diphyllobothrium latum (fish tapeworm) Diphyllobothrium latum is found mainly in cold climates. Hymenolepis nana. pain and weight loss.103). The eggs are lighter in weight and float to the surface where they can be collected (Fig. Ascaris lumbricoides. 10 ml Wooden applicators Gauze Petri dishes 95% Ethanol Ether Willis solution (reagent no.103 Principle of the flotation technique Materials and reagents q q q q q q q q q q q q Microscope Microscope slides Coverslips Wide-mouth bottle.

Parasitology 153 Method Preparation of grease-free coverslips 1. one by one. 5. 3.5 g of stool in a wide-mouth bottle. Mix in a cylinder: 10 ml of 95% ethanol and 10 ml of ether. Place approximately 0. 40 mesh (425 mm). Check that the coverslip is in contact with the liquid.104 Preparation of grease-free coverslips Concentration of parasites 1. Remove the coverslip with care. 4. The above steps are summarized in Fig. 3.4. 4. Pour into a Petri dish and in it place 30 coverslips. Fill the bottle to the 2. Place a coverslip carefully over the mouth of the bottle.2 Formaldehyde–ether sedimentation technique (Allen & Ridley) Principle The stool specimen is treated with formaldehyde. with no air bubbles. 4. Place the coverslip on a slide and examine under the microscope (using the ¥10 objective) at once because the preparation dries very quickly. 4.2 cm diameter (nylon coffee strainers provide an inexpensive alternative) Small porcelain or stainless steel dish or beaker Pasteur pipette q q . Materials and reagents q q q q q q q q q Microscope Microscope slides Coverslips Centrifuge Test-tubes Test-tube rack Centrifuge tubes Wooden applicators Brass wire filter. Fig. Otherwise seal the coverslip with petroleum jelly and wax. 2. shake and leave for 10 minutes. 4. Keep them in a dry Petri dish. crush the portion of stool and mix it well with the solution. Lumpy residues are removed by filtration. Using an applicator. Then fill the bottle to the top with Willis solution. Use the fine adjustment of the microscope to examine every object in the field (eggs tend to stick to the coverslip and are not immediately distinct).104. Fatty elements of the faecal suspension are separated by extraction with ether (or ethyl acetate). Take the coverslips out one by one and dry them with gauze. a drop of liquid should remain on it. Leave for 10 minutes. 7. which sediments any parasites present. 2. which preserves any parasites present.5.5-ml mark with Willis solution. the suspension should be completely uniform. followed by centrifugation.

154 Manual of basic techniques for a health laboratory q Formalin. Using a wooden applicator.5. loosen the fatty plug and discard the supernatant 10. 6. 4. consisting of a conical-based container. a plastic strainer. the steps involving ether should be done outside the cabinet.3 Formaldehyde–detergent sedimentation technique Principle The formaldehyde–detergent sedimentation technique is an inexpensive. 2% solution (prepared by diluting formaldehyde. safe and simple quantitative sedimentation method in which a measured amount of faeces is mixed in formaldehyde–detergent solution of low specific gravity. Allow the fluid remaining on the sides of the tube to drain on to the deposit and then mix well. a beaker and a commercial detergent. q Method Details of the method as supplied with the kit are as follows: 1. transfer a drop on to the slide and cover with a coverslip. Loosen the fatty plug with an applicator and pour the supernatant away by quickly inverting the tube (Fig. Emulsify the faeces in the formalin and filter into the dish. the small amount of fine sediment which forms is examined under the microscope for ova and the eggs are counted to give a quantitative result. 9. 5. The detergent “clears” the faecal debris in a short time. Place the sample in a centrifuge tube containing 7 ml of 10% formalin. If the extraction system of the cabinet is not fireproof. 4. diluted 1:50 with distilled water Formalin. Wash the filter (with soapy water) and discard the lumpy residue. Fig. Materials and reagents q q q Microscope Microscope slides Commercial test kit. 37% solution in 900 ml of distilled water) Ether (or ethyl acetate). Stopper the tube and mix well. Add 3 ml of ether (or ethyl acetate). 4. remove a small amount (approximately 0. Following sedimentation and clearing.105 After centrifuging the suspension. q Method 1. Transfer the resulting suspension back to the centrifuge tube and centrifuge at 2000 g for 1 minute. It is now common practice to perform all the above steps in a biological safety cabinet. The suspension is sieved and is then left undisturbed to allow the ova to sediment under their own weight. 3. 7. Transfer the filtrate to a large test-tube. 10% solution (100 ml of formaldehyde. 4. Use the ¥ 10 and ¥ 40 objectives to examine the whole of the coverslip for ova and cysts. 37% solution 1:50 with distilled water).5 g) of faeces from both the surface and the inside of the stool specimen. a Pasteur pipette. . 8. Ethyl acetate provides a less flammable alternative to ether. 2. Fill the conical-based container to the 10-ml mark with 2% detergent in 2% formalin. Using the pipette.105).

108).107 Removing the supernatant .107).5 ml of fine sediment. Using the plastic strainer. Using the spoon attached to the lid of the container. Rinse the container and then add the filtrate. the emulsified faeces can be transported back to the laboratory for examination. Under field conditions. taking care not to disturb the sediment which has formed in the base of the container (Fig. mix and allow to sediment for a further 1 hour. cysts and larvae of other parasites will be sedimented. transfer approximately 350 mg of faeces to the container and mix well in the formaldehyde–detergent solution. Note: If the supernatant fluid is not removed after 1 hour. 4. Count all the ova present and multiply the number by 3 to give the approximate number per gram of faeces. transfer the entire sediment to a slide and cover with a 22 mm ¥ 40 mm coverslip (supplied with the kit) (Fig.106). 5. 4. Remove and discard the supernatant fluid. Add 10 ml of the formaldehyde–detergent solution. Parasitology 155 2. Using the Pasteur pipette. Further clearing of the faecal debris will take place. 9. using the ¥ 10 objective with the condenser iris closed sufficiently to give good contrast. 4. 3. but instead a further 10 ml of reagent is added and the suspension is remixed and allowed to sediment overnight. The schistosome eggs are fixed and will not become distorted. strain the suspension into the beaker supplied with the kit (Fig. 4.106 Straining the suspension Fig. leaving approximately 0. 7. 6. ova. Carefully remove and discard the supernatant fluid. 4. 4. The technique is of particular value in laboratories without the facilities to perform the Fig.4. Examine the entire preparation under the microscope. Stand the container upright in the rack provided and leave for 1 hour (do not centrifuge). 8.

2.5–3. seal with cellophane tape and keep for 7–8 days at room temperature.108 Transferring the sediment to a slide formaldehyde–ether sedimentation technique. Record the serial number or name of the patient indelibly on the tube. into a test-tube containing filtered or boiled water 2. 3. clean end first. The formalin preserves the parasites without distorting their morphology.156 Manual of basic techniques for a health laboratory Fig. Materials and reagents q q q q q q q Microscope Cellophane tape Test-tubes Test-tube rack Strips of filter-paper (30 mm ¥ 150 mm) Spatula Lugol iodine. 4. Plug the tube with cotton wool or.5. Any larvae of Strongyloides stercoralis present in the specimen migrate against the current of water that rises by capillary action and accumulate at the bottom of the tube. using the ¥ 10 objective.0 cm deep. However.5% solution (reagent no. These must be . Stain with iodine solution for 1 minute and then examine under the microscope. 0. preferably. Method 1. 4. but leave the last 4 or 5 cm clean to be put into water. 4. Put the strip of filter-paper. Look for the larvae at the bottom of the tube.4 Sedimentation technique for larvae of Strongyloides stercoralis (Harada–Mori) Principle A strip of filter-paper is partially submerged in a test-tube containing water. 5. the larvae may have hatched into filariform (infective-stage) larvae. fold the strip at the top so that the bottom does not touch the bottom of the tube. stercoralis. The larvae usually seen in fresh stool specimens are the rhabditiform (first-stage) larvae of S. if the stool was passed more than 12 hours earlier. Use the spatula to spread a small quantity of the faecal specimen along a strip of filter-paper (previously folded lengthwise to keep it straight). 37).

duodenale or N. the use of benzidine is no longer recommended because it has been shown to be carcinogenic.4. 4. q The main distinguishing features of S. it is S. If you see a larva with a long mouth opening and do not see a genital primordium. which may also hatch in stools 12–24 hours after passage. It was originally developed using benzidine.6. The iodine kills the larvae and makes the features easier to see.6 Characteristics of larvae of Strongyloides stercoralis and Ancylostoma duodenale or Necator americanus Larval stage Rhabditiform S. The genital primordium will be more visible in preparations stained with iodine. stercoralis and A. americanus larvae are summarized in Table 4. duodenale or N. However. stercoralis may indicate a systemic hyperinfection. tumours or inflammation. the patient should not: — eat any meat. for example intestinal schistosomiasis.6. — brush his or her teeth vigorously. it is A. — take any drugs containing iron compounds. q If you see a larva with a short mouth opening and a prominent (clearly visible) genital primordium.1 Principle Oxygen is produced when the haemoglobin in blood comes into contact with hydrogen peroxide. or for detection of bleeding in the intestine caused by polyps. You will need to use the ¥ 40 objective to see these structures. stercoralis. 4. Note: For 1 day before the examination. duodenale or N.2 Materials and reagents q q Centrifuge Conical centrifuge tube Table 4. stercoralis Buccal cavity short (4 mm) Oesophagus one-third of body length with 2 swellings Genital primordium large (22 mm) Anal pore 50 mm from posterior end Filariform Size 200–500 mm ¥ 15–20 mm Unsheathed Tail forked or blunt Oesophagus half of body length with no swelling A.6 Chemical test for occult blood in stools This test is used for screening for parasitic infection.6 and illustrated in Fig.109. The liberated oxygen reacts with aminopyrine (aminophenazone) to yield a blue colour. americanus. Parasitology 157 differentiated from larvae of Ancylostoma duodenale and Necator americanus (hookworm). americanus Buccal cavity long (15 mm) Oesophagus one-third of body length with 2 swellings Genital primordium small (7 mm) Anal pore 80 mm from posterior end Size 200–500 mm ¥ 14–20 mm Sheathed Tail tapered Oesophagus one-third of body length with no swelling . 4. The appearance of filariform larvae of S. 4.

2. 10% solution (reagent no. GR: genital rudiment. 4. 4. Decant the supernatant fluid into another test-tube and keep it.110). or until the solids are precipitated (a hand-operated centrifuge can be used).109 Features for the identification of larvae of Strongyloides stercoralis and Ancylostoma duodenale or Necator americanus M: mouth. Note: The glassware used for the test must be clean. 4. Add 7 ml of distilled water and mix thoroughly (Fig. 3.25 g of aminopyrine in the bottom of a test-tube — add 5 ml of 95% ethanol. q q q q q q q q q q Applicators Measuring cylinder. 20 ml Test-tubes Test-tube rack Positive control tube (containing a 1% solution of blood in water) Negative control tube (containing distilled water) Acetic acid.6. 4.5.158 Manual of basic techniques for a health laboratory Fig. .3 Method 1. 2) Hydrogen peroxide (fresh 10% solution) 95% Ethanol Aminopyrine. AP: anal pore. Immediately before carrying out the test. Centrifuge at low speed (1000g) for about 5 minutes. O: oesophagus. prepare a solution of aminopyrine: — put about 0.1). crystalline. with no traces of blood (see section 3. Put a portion of stool (approximately 4 ml) in a centrifuge tube.

and they can be identified in a blood film.4. — responsible for trypanosomiasis. they may also be found in the blood.111). with headache. Meningonema.111 Chemical test for occult blood in stools a: Positive reaction. 4.7. b: negative reaction. — Plasmodium spp. Fig. Elephantiasis of the scrotum. is rare in brugian filariasis (caused by Brugia malayi ). swelling of one leg.4 Results If the reaction is positive a red colour appears between the two layers of liquid (Fig.110 Mixing the stool specimen with distilled water 4. They include: — species belonging to the genera Brugia. Do not mix. Loa. without mixing: — 10 drops of 10% acetic acid solution — 5 ml of the aminopyrine solution.6. — responsible for malaria. can be diagnosed by examination of stained blood specimens under the microscope. and are transmissible between them. Dirofilaria. such as is seen in bancroftian filariasis (caused by Wuchereria bancrofti). the most important is subperiodic Brugia malayi. — Trypanosoma spp. Add 10 drops of the 10% hydrogen peroxide solution. The larvae of the adult worms — the microfilariae — invade the blood. 4. Report the results as follows: — — — — pale red = positive reaction (+) red = strong positive reaction (++) dark red = very strong positive reaction (+++) no change in colour = negative reaction (-). Of these. nausea. . 4. Infection by these parasites and Borrelia spp. Add to the test-tube containing the supernatant fluid. but sometimes migrate to the eyes (which may result in blindness).1 Filariae There are many species of filariae. 4. Fig. Infections among populations in regions where bancroftian and brugian filariasis are endemic may remain asymptomatic. Onchocerca and Wuchereria — responsible for filariasis. 4. hold the tip of the pipette containing the aminopyrine solution against the inside wall of the test-tube and allow the liquid to run down the wall. 6. In advanced cases. Only eight filarial species have adapted to humans. Mansonella. hydrocoele and sterile abscesses. but most are parasites of animals and rarely affect humans. The filarial worms inhabit the lymphatic system. Parasitology 159 5. Let it stand for 1 minute. The results must be read within 5 minutes of adding the hydrogen peroxide solution. elephantiasis of the lower extremities may occur due to obstruction of the lymphatic circulation. To prevent mixing. The microfilariae of Onchocerca volvulus are normally confined to the skin (see below). Attacks of lymphadenopathy lasting several days occur at regular intervals. The main clinical symptoms of lymphatic filariasis are lymphadenopathy and lymphangitis.7 Parasites of the blood and skin Parasites that spend all or part of their life cycle in blood or tissue are known as haemoparasites.

Loa loa. Table 4. To see the highly motile microfilariae. Caribbean Central and West Africa. Central and South America.85% solution (reagent no. it is examined as a wet preparation between a slide and coverslip under the microscope. The initial infection is characterized by a transient. but have been associated with lymphadenopathy.7 shows the geographical distribution of these species. known as a “Calabar swelling”. Materials and reagents q q q q q q q q Microscope Microscope slides Coverslips Pasteur pipette Needle (for intramuscular or subcutaneous injection). Adult worms may migrate across the conjunctiva of the eyes. encephalopathy and cardiomyopathy. The microfilariae develop into infective larvae which invade the mouth parts of the insect. Central and South America Tropical Africa. Non-residents visiting these areas are susceptible to symptomatic infection. 0. but the infection does not cause blindness.7 Geographical distribution of filarial worms Species Brugia malayi Brugia timori Loa loa Mansonella ozzardi Mansonella perstans Onchocerca volvulus Wuchereria bancrofti Asia Parts of Indonesia Central and West Africa Central and South America. Chronic infection may lead to complications such as renal disease. localized. . Examination of skin for microfilariae of Onchocerca volvulus A very small piece of the patient’s skin is collected. abdominal pain.160 Manual of basic techniques for a health laboratory Microfilariae of the following species are found in human blood: Brugia malayi. pruritus. and pains in the knees and ankles. urticaria and Calabar-like swellings (see above). fever. Brugia timori. Mansonella perstans. 22-gauge Scalpel or razor blade Sodium chloride. 53) 70% Ethanol. causing inflammation. Infections with Mansonella ozzardi are also generally asymptomatic. Microfilariae are transmitted by mosquitoes. Infections with Loa loa among populations of areas where it is endemic are often asymptomatic. parts of Arabia Endemic in many tropical countries Geographical distribution 1 Previously known as Dipetalonema perstans. Infections with Mansonella perstans generally seem to be asymptomatic.1 Mansonella ozzardi and Wuchereria bancrofti. but have been associated with pruritus. which feed on the blood of infected humans. flies and midges. Table 4. subcutaneous swelling.

112 Sites for collection of slit skin specimens from patients with nodules 1: chest (over the ribs). see Fig.112): — on the chest (over the ribs) (1). 6.113). 5. Pull the skin away from the flesh with the point of the needle (Fig. — on the back (shoulder-blades) (4). — on the legs (tibia) (3). 4. 4. 3. — the back (centre of the shoulder-blade — 3 in Fig. 4. Place one drop of sodium chloride solution on a slide.114 Lifting the skin with a needle . 2. 4. take specimens from: — the calf (upper outer part — 2 in Fig. 7. two from the calves and two from the shoulder-blades) be examined before reporting a negative result.116). 4. If the patient does not have nodules.114). 3: legs (tibia). Flame the scalpel (or razor blade) and the needle with ethanol. 3: back (shoulder blades). Disinfect the chosen area with a gauze pad dipped in ethanol. — on the hips (2). Take the specimen from the skin in the centre of the nodule.113). Using your left hand.4. 4: back (shoulder blades).113). Cut with a quick stroke the piece of skin pulled up by the point of the needle. 2: hips. pierce the skin with the point of the needle to a depth of 2–3 mm. Parasitology 161 Method Collection of specimens Look for nodules (see Fig. 4. when pushed with the fingertips they slide about under the skin. 2: calves (upper outer part). 4. 4. Fig. The nodules are round and hard. Fig. 1–5 cm in diameter. as close to the needle as possible (Fig. It is recommended that six specimens (two from the buttocks.115). Place the cutting edge of the scalpel or razor blade on the stretched skin above the point of the needle (using your right hand. take the skin specimen from the top of the buttocks (the upper outer part where intramuscular injections are given — 1 in Fig.113 Sites for collection of slit skin specimens from patients without nodules 1: top of the buttocks. If the examination gives a negative result. 1. 4. 4. Fig. 4.

you may see one or two microfilariae moving. it might be damaged. or during mass epidemiological surveys: 1. Collection of specimens in the field If no microscope is available. If any part of the specimen is not in contact with the liquid. Wait 15 minutes for the microfilariae to leave the skin. 4. If no microfilariae emerge. Tail: curved and tapered.162 Manual of basic techniques for a health laboratory The specimen should be about this size: q (2–3 mm in diameter). shake the bottle well. Apply an adhesive dressing.115 Place the blade above the point of the needle 9.117). 8. if only one microfilaria is present. Cover it with a coverslip and examine it under the microscope. Fix the specimen by adding 2 ml of 10% formaldehyde solution (reagent no. Fig. 2. 4. Wait 2–3 minutes. Fig. injecting it under the coverslip with a Pasteur pipette. Place the piece of skin in a small bottle containing 2 ml of sodium chloride solution. wait for 10 minutes and look at the centre of the piece of skin. as the microfilariae can be identified by their characteristic angular curves. 28). Do not flatten the piece of skin. Meanwhile. If you are in any doubt. they will be clearly visible. The specimen should not be bloodstained.117 Onchocerca volvulus microfilariae in wet preparation Length: 200–315 mm. It should remain attached to the tip of the needle. 4. If any are present. Centrifuge the liquid (after removing the piece of skin) at medium speed (2000g) for 5 minutes. Curvature of the body: angular. 10. Microfilariae are highly motile. . Place the fragment of skin in the drop of sodium chloride solution on the slide (using the scalpel or razor blade if necessary). they will be seen moving towards the sodium chloride solution (Fig. Cover with a coverslip. Microscopic examination Examine the wet preparation under the microscope using the ¥ 10 objective. add more solution. take a fresh blood specimen from the patient’s finger and prepare a smear on a slide. the biopsy must be bloodless. Mix and replace the cap on the bottle. 4. Microfilariae of Onchocerca volvulus have the following features: Fig. Width: 5–9 mm (the same as an erythrocyte). Front end: slightly broader than an erythrocyte. Transfer the deposit from the centrifuge tube to a slide and cover it with a coverslip. If you see any microfilariae. prepare a stained skin smear (see below) and a stained thick blood film (see page 170) to identify the species. 4. 5.116 Collecting a slit skin specimen 3. clean the spot from which the specimen was taken with ethanol. When you return to the laboratory. If microfilariae are present. Staining is not necessary. until the whole area underneath the coverslip is wet.

118): — they have no sheath. 4. Recommended procedures for the detection and identification of microfilariae in blood The microfilariae of some species (e. Loa loa) and the most common strain of Brugia malayi appear in the blood with a marked nocturnal or diurnal periodicity (Table 4.8). — the tail ends in a rounded crook. Its microfilariae are found in the skin and differ from Onchocerca volvulus in the following ways (Fig. malayi do not show the same degree of periodicity. Table 4. Mansonella ozzardi ). 4. Stained microfilariae of Onchocerca volvulus show the following features (Fig. — the front end is broad. Other microfilariae found in skin biopsies Mansonella streptocerca causes an itchy dermatitis of the infected area. Fig. — the nuclei are large and oval and stain blue-black.119 Mansonella streptocerca microfilaria in wet preparation . 4. Parasitology 163 Procedure for obtaining a stained specimen Examine the deposit using the ¥10 objective. they are well separated and do not extend to the tip of the tail. A smear is made on a slide by crushing the skin specimen. — the nuclei are smaller and reach the tip of the tail. — the tail tapers gradually and ends in a sharp curve.9 shows the recommended times for collecting blood specimens for testing for periodic and subperiodic species of microfilariae. The times for collection of blood specimens should be selected in accordance with the patient’s clinical symptoms and travel history.g. Wuchereria bancrofti ). they are nocturnally subperiodic or diurnally subperiodic (e.118 Onchocerca volvulus microfilaria in Giemsa-stained smear Fig.g. 4. Other species show no periodicity (e.4. — the body shows rigid curves.g. It is fixed using methanol and stained with Giemsa stain (see page 170). — the front end is not broad.119): — they are slightly shorter (180–240 mm). Other species and strains of B. — they are less broad (5–6 mm — half the width of an erythrocyte).

Direct finger-prick samples may give adequate results in areas where filariasis is endemic. Fig. Prick with the lancet. 4. 5. 59) or heparin anticoagulant. 0. Add an equal-sized drop of sodium chloride solution to the slide. An absolute minimum of one “day blood” specimen (taken around 13:00) and one “night blood” specimen (taken around 24:00) should be examined. This is usually sufficient to detect mixed infections and infections with subperiodic strains. Method 1. Examine the smear systematically under the microscope using the ¥ 10 objective with the condenser aperture reduced. 4. Dry well. A blood sample for microfilariae is best examined immediately. 4. If a “night blood” sample will not be examined until the following morning.85% solution (reagent no. 53) 70% Ethanol.120 Collecting a capillary blood sample . all pathological specimens should be treated as potentially hazardous.120). 3. 2. Collect the first drop of blood that appears (it contains most microfilariae) directly on to the middle of the slide (Fig. Sterilize the third finger with ethanol.164 Manual of basic techniques for a health laboratory Note: Although microfilariae are not directly infectious to humans. To identify the species of microfilariae. 6. leave it at room temperature. Microscopic examination of capillary blood q q q q q q q Materials and reagents Microscope Microscope slides Coverslips Blood lancets Cotton wool swabs Sodium chloride. For each specimen. collect 5–10 ml of blood into a 2% solution of trisodium citrate in saline (reagent no. Cover the preparation with a coverslip. prepare two smears on another slide using two more drops of blood and stain them as described on page 170. Mix the blood and sodium chloride solution using the corner of a slide. The first sign of the presence of microfilariae is rapid movement among the erythrocytes.

. pointed.0–5.0–9. hooked tail filled with nuclei.) Characteristic B.Table 4. South America Biting midges (Culicoides spp. 180–225 in 2% formalin 4. Indian subcontinent Lesser Sunda islands. aperiodic 230–250 in smears.) Central and West Africa.0 4. malayi B. long slender tail.0 Bluntly rounded. streptocerca O. subterminal and terminal nuclei widely separated Tapered.8 Tail Tapered.0 Tapered. timori 4. terminal and subterminal nuclei Small size. 330–385 in 2% formalin Width (mm) 5. nuclei irregularly spaced Nocturnala Nocturnal Morphology of microfilariae Absent 300–315 in skin snips Present 240–300 in smears. orbit Blood Diurnal Adults Lymphatic system Lymphatic system Microfilariae Blood Blood Periodicity of microfilariae Present Absent Absent Absent 180–240 in skin snips 190–200 in smears. dispersed nuclei a b Nocturnally subperiodic in Indonesia. subterminal and terminal nuclei widely separated Key features Sheath unstained in Giemsa.0 Long. single row of nuclei to end of tail 165 Long head space.) Subcutaneous tissues Mesentery Dermis Subcutaneous tissues Skin NA Skin NA Blood Aperiodic Blood Aperiodic Central and West Africa Tabanid flies (Chrysops spp.5–10.) Habitat Subcutaneous tissues.) Lymphatic system Blood Nocturnalb Africa. Malaysia.0 Tapered. blunt tail filled with nuclei. ozzardi M.0 Bluntly rounded. Central and South America Blackflies (Simulium spp. loa M. Timor Vectors Mosquitoes (Anopheles and Mansonia spp. nocturnally subperiodic in rural areas of Thailand. Anopheles and Mansonia spp.) Central and West Africa Biting midges (Culicoides spp. sheath unstained in Giemsa. 205–250 in 2% formalin 3.4–6. Parasitology Geographical distribution South-east Asia. slender. Caribbean Biting midges (Culicoides spp. body in smooth curves. nuclei to end 160–205 in smears. bancrofti Tropical and subtropical countries Mosquitoes (Culex.) and blackflies (Simulium spp. Aedes. perstans M. 240–300 in 2% formalin 265–325 in smears.0–7. aperiodic Slender shape. anucleate 7.0 Typically flexed.) Central and South America.0–5. nuclei to end 5. parts of the Philippines and Thailand. sheath unstained in Giemsa. Diurnally subperiodic in New Caledonia and Polynesia. 270–300 in 2% formalin 5. volvulus W. tapered to point. occur in skin and occasionally in urine or blood after treatment Short head space. anucleate Small size. occur in skin Flexed tail.0–6. sheath stains pink in Giemsa.) Mosquitoes (Anopheles spp.8 Characteristics of common human filarial parasites L. bent into a hook.0–6. 275–320 in 2% formalin 5. anucleate Sheath Present Present Length (mm) 175–230 in smears. terminal and subterminal nuclei Long head space.

e.122 A microfilaria of doubtful pathogenicity Length: about 150 mm. 2% solution (prepared by diluting 37% formaldehyde solution 1:50 with distilled water) or saponin. 4. 48) Ether 70% Ethanol.122). Mansonella ozzardi. Microscopic examination of venous blood concentrated by centrifugation q q q q q q q q q q Materials and reagents Microscope Microscope slides Syringes (5 ml or 10 ml) Needles for venepuncture Centrifuge or microhaematocrit centrifuge Conical centrifuge tubes or microhaematocrit capillary tubes Plastic modelling clay Adhesive tape Anticoagulant: trisodium citrate.121 and 4.g. e. perstans.121 A pathogenic microfilaria Length: 250–300 mm. 1% solution (reagent no. to gain some indication of the species seen and its pathogenicity from a fresh smear (Figs. Fig. It is possible. M. Wuchereria bancrofti. Staining is generally required to identify microfilariae in blood smears. Brugia malayi. thickness: about 4 mm (half the diameter of an erythrocyte).8. 59) Formalin.166 Manual of basic techniques for a health laboratory Table 4. Fig. however. 2% solution in saline (reagent no.g. Loa loa. q q . thickness: 6–8 mm (diameter of an erythrocyte). 4.9 Recommended times for collection of blood specimens for testing for microfilariae Speciesa Periodic (nocturnal) Periodic (diurnal) Subperiodic (nocturnal) Subperiodic (diurnal) Aperiodic a Recommended collection time 23:00–01:00 (peak 24:00) 12:00–14:00 (peak 13:00) 20:00–22:00 (peak 21:00) 15:00–17:00 (peak 16:00) any time (day or night) See Table 4. 4.

using the ¥ 10 objective with the condenser aperture reduced. just above the layer of leukocytes and erythrocytes (Fig. 4. . Expel into a bottle containing 1 ml of trisodium citrate solution. Pour off the supernatant fluid. Collect two drops of capillary blood from the finger on to a slide and mix with one drop of 2% trisodium citrate solution. Lay the capillary tube on a slide and secure the two ends with adhesive tape. 3. Centrifuge for 5 minutes at 10 000g. 4. 5.124). 5. Motile microfilariae will be seen at the bottom of the column of plasma. 4.124 Motile microfilariae E: erythrocytes. Spread the drop to form a thin smear and leave to air-dry. Fig. 3. 2. Mix. Place one drop of the deposit on a slide. Examine the dividing line between the blood cells and the plasma under the microscope (Fig. P: plasma. Measure into a conical centrifuge tube 10 ml of 2% formaldehyde solution. Seal one end of the tube with plastic modelling clay or by heating. 4. Alternative method using a microhaematocrit centrifuge 1. Parasitology 167 Method 1. Three-quarters fill a microhaematocrit capillary tube with the citrated blood. The tube can be snapped at the bottom of the column of plasma (see Fig. Mix. 4. then stain immediately as described on page 170 to identify the species of microfilaria.4. Collect 4 ml of venous blood. 4. 4. Use the first drop from each piece of the broken tube to prepare a thick film. Mix. Collect 4 ml of venous blood. Expel into a bottle containing 1 ml of trisodium citrate solution. Fix the smear using a 1:1 mixture of ethanol and ether.124).123 Examining the microhaematocrit capillary tube under the microscope Fig. Tap the tube to mix the deposit.123). Stain the film as described on page 170 to identify the species. Capillary blood can also be examined by this method. Add 1 ml of citrated blood. 2. Centrifuge in a microhaematocrit centrifuge at 10 000g for 2 minutes. Wait 5 minutes for the erythrocytes to lyse. L: leukocytes. Leave to dry for 2 minutes.

Moisten the filter-paper pad with a few drops of distilled water and cover with the membrane filter. 4. 3. It is recommended that identification be performed on stained preparations (see page 170). 6.127).168 Manual of basic techniques for a health laboratory Alternative method using saponin lysing solution 1. Remove the syringe from the filter holder (taking care to avoid disturbing the filter) and draw up 10 ml of distilled water.125). 4. with lid Blunt forceps Sodium chloride. Draw up 10 ml of distilled water into a syringe. 2. Wait for 2–3 minutes. Place the filter on the filter holder. Gently push the blood through the filter into a dish containing disinfectant solution (Fig. Mix the blood gently and leave for 15 minutes to allow the erythrocytes to lyse. 1 In areas endemic for Mansonella perstans. 53) Absolute methanol Distilled water. .) 7. Rotate gently to mix the contents. Transfer the deposit to a slide and cover with a coverslip. (Microfilariae will still be motile in a “night blood” sample examined the following morning. Add 10 ml of citrated blood (see above) to 10 ml of saponin lysing solution. Microscopic examination of venous blood concentrated by filtration q q q q q q q q q q q q Materials and reagents Microscope Microscope slides Coverslips Syringe. 6. Centrifuge at 2000g for 15 minutes. 4. a membrane filter with a pore size of 3 mm should be used. Considerable experience is required to identify unstained microfilariae. Connect the syringe to the filter holder. for the erythrocytes to lyse. 0. 4. 4. Count the number of microfilariae in the preparation and divide by 10 to give the number of microfilariae per ml of blood. 2. Reconnect the syringe to the filter holder and gently push the water through the filter into the dish containing disinfectant solution. Examine the entire deposit for motile microfilariae using the ¥ 10 objective. Method 1. 15 ml Swinnex-type filter holder Polycarbonate membrane filter (25 mm diameter. Remove the supernatant with a pipette and discard it into a dish containing disinfectant.126). to remove the debris from the filter (Fig. 3. 5. 15 ml.85% solution (reagent no. 5. 5 mm pore size)1 Filter-paper pad (25 mm diameter) Shallow dish. Draw 1 ml of fresh blood or citrated blood into the syringe (Fig.

using the ¥ 10 objective. Dismantle the filter holder and remove the membrane filter using forceps. Place the membrane filter. Add a drop of sodium chloride solution and cover with a coverslip. 9. follow the method described above.125 Drawing up citrated blood into a syringe Fig. (Microfilariae will still be motile in a “night blood” specimen examined the following morning. with the following modifications: Fig. 4. Discard the disinfectant solution into a sink.126 Filtering the blood sample Fig. top side facing up. Reconnect the syringe to the filter holder and push the air through the filter over the dish containing disinfectant. 10. on a slide. to remove excess water from the filter. To prepare a stained preparation. 12. Remove the syringe from the filter holder and draw up approximately 5 ml of air. Examine the entire membrane for motile microfilariae. It is recommended that identification be performed on stained preparations (see below). 11. 4. Count the number of microfilariae in the preparation and divide by 10 to give the approximate number of microfilariae per ml of blood. 8. Parasitology 169 7.127 Rinsing the filter . 4. Considerable experience is required to identify unstained microfilariae. Remove the syringe from the filter holder.4.

often enclosed in a sheath and filled with the nuclei of many cells (Fig. 10. Examine the preparation under the microscope. Use the ¥ 10 objective first to locate the microfilariae. the sheath may extend a short or long distance beyond either extremity. respectively. Technique for staining microfilariae q q q q q q Materials and reagents Microscope Microscope slides Giemsa stain (reagent no.8. 5. 12.8) for 5 minutes. then identify the filarial species using the ¥ 40 and ¥ 100 objectives.8) for 30 minutes. Results Under the light microscope microfilariae appear (after appropriate staining) as primitive organisms.170 Manual of basic techniques for a health laboratory 8. on a slide. (This second stain is required because Giemsa stain alone does not stain the sheath of Loa loa very well. Method 1. depending on the stain used. return the slide to the Giemsa stain solution for another 5–10 seconds. In some species. Remove the syringe from the filter holder and draw up approximately 7 ml of air and 3 ml of methanol. pH 6. 4. the sheath displays a unique staining quality which aids in species identification. to fix the microfilariae and remove excess methanol from the filter. The anterior extremity is charac- . Reconnect the syringe to the filter holder and push the air through the filter over the dish containing disinfectant. Not all species have a sheath.) 6. Dismantle the filter holder and remove the membrane filter using forceps. Examine the preparation under the microscope using the ¥ 10 objective. 13. Allow to air-dry. Place the membrane filter. 29) Delafield’s haematoxylin stain (reagent no. Wash in buffered water. 19) Methanol Buffered water (reagent no. Reconnect the syringe to the filter holder and push the methanol and air through the filter over the dish containing disinfectant. to remove excess water from the filter. Allow the smear to air-dry. Prepare a thick blood smear of the deposit as described on page 174. 4.128). pH 6. 3. Stain with Delafield’s haematoxylin stain (diluted 1 in 10 with buffered water. Remove the syringe from the filter holder. pH 6. The nuclei of the cells which fill the body are usually darkly stained and may be crowded together or dispersed (see Fig. Stain with Giemsa stain as for thick films (see page 175) and examine the entire filter membrane using the ¥ 10 objective. 9. Stain with Giemsa stain (diluted 1 in 20 with buffered water. If it is difficult to distinguish the nuclei of the microfilariae. 4. 2. serpentine in shape. 15). In those that do. 11.128). top side facing up. Fix in methanol for 1 minute.

As you look from the anterior to the posterior end of the body you will see additional spaces and cells that serve as anatomical landmarks. cephalic space twice as long as broad Tail uniform. cephalic space as long as broad Nuclei extending to tip of tail. R2. but on all the features taken together. q who 90589 . At other times. teristically devoid of nuclei and is called the cephalic or head space. Parasitology 171 Cephalic space With a sheath Sheath Anal pore R1 cell Without a sheath R4 cell R3 cell R2 cell Nerve ring Excretory pore Excretory cell Microfilariae In blood In skin With sheath Without sheath Without sheath Nuclei extending to tip of tail Nuclei not extending to tip of tail. small. small. hooked tail Nuclei not extending to tip of tail.4. The identification must not be based on a single characteristic. Table 4. before deciding on their species. it should be possible to identify with certainty the species observed. Sometimes it is difficult to see the sheath. Other useful features include the shape of the tail and the presence or absence of nuclei within it. cephalic space as long as broad Brugia malayi Loa loa Wuchereria bancrofti Mansonella perstans Mansonella ozzardi Mansonella streptocerca Onchocerca volvulus Fig.8 summarizes the features of common human filarial parasites that are used in their identification. Some of these structures and their positions are useful in identifying the species. Identification of species can be difficult and mistakes are frequently made. R4: rectal cells. In some species an amorphous mass called the inner body and four small cells (known as rectal cells) can be seen. excretory cell and anal pore. If a systematic study is made of all the characteristics mentioned above. it may be short or long. It is good practice to examine several microfilariae carefully. thin filaria. tail blunt Nuclei not extending to tip of tail. These include the nerve ring.128 Microfilariae found in humans R1. R3. Note: q Sometimes the microfilariae of the periodic strain of Brugia malayi lose their sheath. thin filaria Nuclei extending to tip of tail. The guidelines for the identification of microfilariae given above and those that appear in most textbooks make identification seem deceptively simple. excretory pore. thick filaria Tail swollen with 2 distinct nuclei. 4. the nuclei do not appear in their characteristic position at the tip of the tail.

Examination of thin films. The sheath is sometimes torn or almost colourless. India. If the patient is from: — Cameroon. The sporozoites travel through the blood to the liver. vivax. — Guyana. — Thailand. 250 mm). These symptoms are often misinterpreted as being the result of a viral influenza infection. Identification of microfilariae in stained thin films is not recommended. the clinical symptoms may be more moderate. It is transmitted to humans through the inoculation of Plasmodium sporozoites by female Anopheles mosquitoes or by blood transfusion. These begin to rupture after 5–20 days. the sheath appears as a colourless space between the tail and the blood cells. P. the parasite is probably Loa loa. If high temperatures are accompanied by mental disturbances marked by hallucinations and cerebral excitation. periodic attacks of high fever and shivering. 4. Always bear in mind where the patient is from or which countries the patient has visited recently. Fig. . distorted and difficult to recognize. If it is damaged when the smear (or film) is being made. the parasite is probably Wuchereria bancrofti. — Ghana. malariae. muscle aches and malaise.2 Plasmodium spp. In Loa loa. The influenza-like symptoms are followed by recurrent.172 Manual of basic techniques for a health laboratory Possible causes of misidentification Broken or folded tail. Some Mansonella perstans are very long (e. Malaria. the parasite is probably Brugia malayi. The replication cycle is repeated at regular intervals. In areas in which malaria is endemic and where the population has developed partial immunity to the disease. and the released merozoites invade circulating erythrocytes. for example. according to the species. Clinical symptoms The first clinical symptoms of infection are low-grade fever. the parasite is probably Mansonella ozzardi.7.g.129 Possible cause of misidentification of Wuchereria bancrofti: broken or folded tail Geographical origin of the patient. it appears to have nuclei extending to the tip like Loa loa. and some Wuchereria bancrofti and Loa loa are small (e. ovale and P. Senegal or the West Indies. where they transform into large tissue schizonts containing considerable numbers of merozoites (tissue schizogony). the microfilariae are shrunken. Wuchereria bancrofti may appear twisted and Loa loa may show a few curves. 4. falciparum. 200 mm). which is often fatal. Badly made smears (or films). 4. is the most important parasitic disease in tropical countries. Unusually large or small microfilariae. eastern Nigeria or the Democratic Republic of the Congo river basin. Plasmodium species infective to humans There are four different species of Plasmodium infective to humans.g. P. headaches. Torn or colourless sheath. this may indicate cerebral malaria.129). If the tail of Wuchereria bancrofti is broken or folded over (Fig. These are P. which is caused by infection with protozoa of the genus Plasmodium.

4. vivax Rare Common Predominant Very rare Predominant Predominant Predominant Predominant Predominant Common Common Common Common The geographical distribution of these species is summarized in Table 4. They may also be detected using an immunological procedure known as a dipstick test (see section 11. Do not mistake thrombocytes superimposed upon erythrocytes for malaria parasites. Identification of Plasmodium spp. The most suitable time for collection is at the height of an episode of fever. Preparation of a thick and a thin blood film on the same slide For routine malaria microscopy. Parasitology 173 Table 4. q q q q q q q Method Blood to be examined for malaria parasites is usually collected at a health centre.5. The thick film is used for the detection of parasites. when the parasites are most numerous in the blood. always report the presence of any malaria parasites you see.10.1) Sterile blood lancets Cotton wool Grease pencil Methanol 70% Ethanol. Blood specimens should always be collected before antimalarial drugs are given. ovale Rare Rare Absent Rare Absent Absent Absent Absent Very rare Rare Very rare Rare Rare P. . falciparum Predominant Predominant Very rare Predominant Common Common Common Very rare Common Predominant Predominant Predominant Predominant P. It is important for the prognosis and treatment of the disease that the species involved are identified in the laboratory. Indian Ocean Pacific Islands P.10 Geographical distribution of Plasmodium spp. infective to humans Country or area Central Africa East Africa North Africa West Africa Central America South America Central and south-west Asia South-east Europe Indian subcontinent Indochina Indonesia Madagascar. If you cannot identify the species. while the thin film is used in identifying the species of parasite. Materials and reagents Microscope Clean glass microscope slides (see section 3.9). in blood films Malaria parasites are usually detected in blood films stained with Field or Giemsa stains. malariae Rare Rare Very rare Rare Rare Common Common Very rare Rare Rare Very rare Rare Very rare P. a thin and a thick film are made on the same slide.

130 Cleaning the finger before collecting a capillary blood sample Fig.130). Using another clean slide as a “spreader”.174 Manual of basic techniques for a health laboratory Fig.133).134). Always handle slides by the edges. 4. 2. using a quick rolling action. 5. .) Use cotton wool lightly soaked in ethanol to clean the finger — using firm strokes to remove dirt and grease from the ball of the finger (Fig. 4. away from the largest drops. firm surface. about this size q.135). Thin film. keeping the spreader at an angle of 45° (Fig.132). dust and extreme heat. Make sure that no strands of cotton wool remain on the finger. With a sterile lancet. level position protected from flies.131 Using a lancet to puncture the ball of the finger 1.131). Label the dry film with a grease pencil by writing across the thicker portion of the thin film the patient’s name or number and date (as shown in Fig. 3. 4. Make sure that the spreader is in even contact with the surface of the slide all the time the blood is being spread. This is for the thin film. select the third or fourth finger. • Apply further pressure to express more blood and collect two or three larger drops. 6. Thick film. 4. Firmly push the spreader along the slide. collect the blood as follows: • Apply gentle pressure to the finger and collect a single small drop of blood. With the patient’s left hand palm upwards. Allow the thick film to dry in a flat. about this size q. quickly join the larger drops of blood and spread them to make an even. 4. 4. touch the small drop with the spreader and allow the blood to run along its edge. Working quickly and handling clean slides only by the edges. on to the slide about 1 cm from the drop intended for the thin film (see Fig. and with the slide with the blood drops resting on a flat. on to the middle of the slide. 4. (The big toe can be used with infants. to make the thick film as follows: Using the corner of the spreader. Dry the finger with a clean piece of cotton wool (or lint). express the first drop of blood and wipe it away with dry cotton wool. Wipe the remaining blood away with cotton wool. By applying gentle pressure to the finger. The thumb should never be used for adults or children. puncture the ball of the finger (Fig. or by a corner. thick film (Fig. 4. 4.

50 and 100 ml Beakers. All that remain are the parasites and the leukocytes.4. 10. 4.133 Preparing a thin blood film Fig. Materials and reagents Microscope Measuring cylinders. the haemoglobin in the erythrocytes dissolves (dehaemoglobinization) and is removed by the water in the staining solution. which can be seen under the microscope. 4.134 Preparing a thick blood film Fig. 4.135 Labelling the slide Staining blood films with Giemsa stain Principle During staining of the blood film. Parasitology 175 Fig. 50 and 250 ml Staining troughs Glass rods Wash bottle Slide forceps Slide racks q q q q q q q q . 4.132 Collecting the blood sample Fig.

Pour the water off. good-quality staining of the thick film is of primary importance. Fig. Pour clean water gently into the trough to remove the deposit on the surface of the staining solution (Fig. 1. until all the slides are totally covered. therefore avoid exposure of the thick film to methanol or methanol vapour. for optimum staining. Using forceps. in sufficient quantity to fill the number of staining troughs being used.138). pH 7. To permit dehaemoglobinization. or by dipping it into a container of methanol for a few seconds. 4.176 Manual of basic techniques for a health laboratory q q q q Timer Giemsa stain (reagent no. making sure that the film does not touch the slide rack. Place them in a slide rack to drain and dry. Pour the stain gently into the staining trough.137). Prepare a 3% Giemsa solution in buffered or distilled water. Mix the stain well. 4. and rinse again in clean water for a few seconds. Gently pour off the remaining stain (Fig.2 (reagent no. With prolonged fixation it may be difficult to detect Schüffner’s dots and Maurer’s clefts. film side downwards.136 Placing the slides in a staining trough 5. 15) or distilled water. Fix the thin film by adding three drops of methanol. .137 Pouring clean water into the staining trough to remove the deposit Fig. 4. 4. 2. Routine method for staining thick and thin blood films Ideally. pH 7.136). 4. 7. in such cases it must be used on the same day. 29) Methanol in a drop bottle Buffered water. This method is suitable for staining 20 or more slides. 6. the thick film should not be fixed. 4. This is often not possible and thick and thin films are generally made on the same slide. Rapid method for staining thick and thin blood films This method is suitable for rapid staining of thick films when urgent results are required. 3. Fig. 4. remove the slides one by one. When this is done. It uses much more stain than the regular method.138 Pouring off the remaining stain In some laboratories with limited supplies the diluted Giemsa stain is reused. Stain for 30–45 minutes out of sunlight.2. Best results are obtained if the blood films have dried overnight. thick and thin films should be made on separate slides. place the slides back to back in a staining trough (Fig. Using forceps.

or by dipping it into a container of methanol for a few seconds. Stain for 5–10 minutes. q q q q q q Method for staining thick films 1. 4. otherwise the thick film will be heat-fixed. Dip the slide into a jar containing Field stain B solution for 3 seconds. Fix the thin film by adding three drops of methanol. Care should be taken to avoid overheating. 5.2. Using a pipette.2 (reagent no. Mix the stain well with a glass rod. Immediately add an equal volume of Field stain A solution and mix well by tilting the slide. therefore avoid exposure of the thick film to methanol or methanol vapour. Staining blood films with Field stain Staining with Field stain allows rapid detection of malaria parasites (but it does not always stain Schüffner’s dots). Place the slide upright in a slide rack to air-dry. three drops of stain per ml of buffered water will give the correct concentration of Giemsa solution. Materials and reagents Microscope Glass jars Slide racks Methanol Field stain (reagent no. or briefly exposing the slide to gentle heat such as that from a microscope lamp. 6. Gently pour the stain on to the slides or use a pipette. 3. Wash off the methanol with buffered water. Parasitology 177 1. the thick film should not be fixed. if a small quantity is being used. One slide requires about 3 ml of madeup stain. if results are required urgently. Place the slide upright in a slide rack to air-dry.4. 2. 15). Wash gently by dipping (once) into a jar of clean water for 5 seconds. 7. 4. To permit dehaemoglobinization. 25) Buffered water. film side downwards. Fix the film in methanol for 1 minute. Prepare a 10% Giemsa solution in buffered or distilled water. pH 7. cover the film with diluted Field stain B (one volume of stain plus four volumes of buffered water). 2. . making sure that the film does not touch the slide rack. Place the slides in the slide rack to drain and dry. Allow to stain for 1 minute. 3. Method for staining thin films 1. Wash off the stain with clean water. Do not tip off the stain and then wash. Dip the unfixed film into a jar containing Field stain A solution for 3 seconds. drying may be hastened by fanning. 5. 2. 6. 5. as this will leave a deposit of scum over the smears. 4. Gently flush the stain off the slides by adding drops of clean water. pH 7. Wash the slide gently as in step 2. Allow the thick film to dry thoroughly. 3.

the infected erythrocytes may remain unchanged or have a different colour or shape. 4. 4.139 Stages of development of malaria parasites Fig.140 Malaria parasites containing pigment . 4. Malaria parasites found in the blood are at different stages of development (Fig. or may contain pink (“Schüffner’s”) or red (“James”) Fig.178 Manual of basic techniques for a health laboratory Microscopic examination Examine the slide under the microscope using the ¥ 100 objective. Thin blood films In thin blood films. Some malaria parasites have granules of pigments in their cytoplasm (Fig. 4.140).139).

vivax P. a few large pink granules (“Maurer’s clefts”) can be found.139) Trophozoites and/or gametocytes. Two methods can be used to count malaria parasites in thick blood films: determination of the number of parasites per microlitre (ml) of blood. Thick blood films are used for estimating the parasite density. as the infected erythrocytes are lysed. Thick blood films In thick blood films. . dots (see Table 4.11 Comparison of infected erythrocytes in thin blood films P.4. monocytes may be seen in the thin blood film. The malaria parasites should have deep red chromatin and blue or pale purplish-blue cytoplasm. with torn jagged edges Dots in the infected erythrocyte Often nonea Stages found (see Fig. In patients who have recently received an injection of an antimalarial drug. falciparum Size of young trophozoite in comparison with diameter of an erythrocyte (at the same stage of development) Appearance of infected erythrocyte P. Parasite density The parasite density is the number of parasites counted in each microscope field. 4.12). the background should be clean and free from debris. Parasitology 179 Table 4. Thin films can be used to identify the species of malaria parasite (see Table 4. Note: In patients who have been suffering from malaria for a long time. many trophozoites can be found in one cell None All stages found in the same film Small pink dots (Schüffner’s dots) All stages found in the same film Large red dots (James dots) always present All stages found in the same film a In some erythrocytes infected with adult trophozoites of P. the cytoplasm often contains brown or greenishblack bodies (siderophils). and the plus system. oval. malariae P. falciparum. ovale One-fifth to onethird of diameter One-quarter to twothirds of diameter One-quarter to twothirds of diameter One-quarter to twothirds of diameter Remains unchanged Remains unchanged or becomes smaller and sometimes more deeply coloured Enlarged and often pale-staining Enlarged. the parasites stain poorly and appear distorted and indistinct. In thick films stained with Giemsa. Schüffner’s dots may be seen around the malaria parasites. the nuclei of leukocytes should be stained dark purple.11). as described below. It usually varies according to the species.

dark blue. dense. Adults living in endemic areas often develop immunity to the disease and have a low parasite density. fine. compact. or (b) in band form (in thin films only) Chromatin: a round dot or red band Mature trophozoite Cytoplasm: rather thin. the blood film is examined for the presence of parasite species and their stages of development.12 Identification of Plasmodium spp. Parasite densityb 1. Determination of the number of parasites/ml of blood is accomplished by counting the number of parasites in relation to a standard number of leukocytes/ml (8000). irregular in size and shape Often very high density Normal in size and shape No red dots usually seen Low density a b The identity of P. with many black particles of pigment. Using two hand tally counters. The parasite density in any area depends mainly on whether the malaria is seasonal or endemic. oval or rounded Colour: pale blue (male) or dense blue (female) Nucleus: one round spot of red chromatin against one edge Pigment: large black granules in the cytoplasm Gametocyte Colour: pale blue (male) or dense blue (female) Nucleus: reddish-pink Pigment: a few blue–black granules in the centre of the cytoplasm or scattered through it Erythrocytes Normal in size May show crenation cells containing mature trophozoites. often contain a few red dots. pale blue ring Chromatin: one or two small red dots (Stage frequently found) (Stage frequently found) Cytoplasm: either (a) round. infective to humans in blood films P. or shaped like a comma or an exclamation mark Chromatin: one or two mediumsized red dots (Very rare) Hardly ever found in blood films (except in very serious cases) Merozoites: 18–32 Pigment: dark brownish-black (Fairly frequently found) Merozoites: 8–10 large red granules surrounded by pale cytoplasm and arranged irregularly (young form) or in a rosette Pigment: always seen Schizont (Fairly frequently found) Shape: similar to a banana or sickle (Fairly frequently found) Shape: large. follow one of these two procedures: . one for counting leukocytes and the other for parasites. blue ring with some granules of black pigment Chromatin: one large red dot Young trophozoite Cytoplasm: small. falciparum (Stage frequently found) P.180 Manual of basic techniques for a health laboratory Table 4. blue ring. malariae (Stage frequently found) Cytoplasm: thick. ovale must be confirmed by examination of a thin blood film. Initially.

very blue with a few particles of brown pigment Chromatin: one large red dot (Quite frequently found) Merozoites: 12–18 large compact red granules surrounded by pale blue cytoplasm Schizont Merozoites: 8–14 large red granules in a rosette. record the results on the record form in terms of the number of parasites/200 leukocytes. after counting 200 leukocytes. ovalea Cytoplasm: regular. round a central mass of particles of brown pigment (Frequently found) Female: oval or rounded. Parasitology 181 Table 4. often pale-staining Schüffner’s dots.) P. the number of parasites is 9 or fewer. malariae by the presence of Schüffner’s dots Gametocyte Erythrocytes Enlarged.11 May appear oval with jagged ends Easily seen large red James dots Medium density Medium density Parasite densityb (i) (ii) if. after counting 200 leukocytes. blue. pale blue A round central pale red nucleus. many particles of orange pigment in the cytoplasm Male: rounded.12 (cont. irregular (sometimes divided into 2–4). often at one end. continue counting until you reach 500 leukocytes and then record the number of parasites/500 leukocytes. some particles of orange pigment in the cytoplasm Shape: large. especially around mature trophozoites See Table 4. small particles of brownishorange pigment Chromatin: 1 red dot Cytoplasm: round.4. compact. dense blue Nucleus: one round red spot Pigment: a few brown particles in the cytoplasm Differentiated from: — P. dense blue A dense red triangular nucleus. quite thick ring Chromatin: one large red dot (Not frequently found) Mature trophozoite Cytoplasm: large. if. . vivax by its brown pigment — P. 10 or more parasites are found. vivax (Stage frequently found) P. irregular. oval or round. dense blue ring Chromatin: one mediumsized red dot Young trophozoite Cytoplasm: blue.

4. multiplying the number of parasites by 8000 and then dividing this figure by the number of leukocytes (200 or 500). P. The result is the number of parasites/ml of blood. It occurs in southern and western Africa. Blood films containing P. where it is called Chagas disease. Reporting results If the result of the examination of the stained blood films is positive. a code of between one and four plus signs is used: + ++ +++ ++++ = 1–10 parasites per 100 thick film fields = 11–100 parasites per 100 thick film fields = 1–10 parasites per single thick film field = more than 10 parasites per single thick film field. Note: Patients with very high parasite densities (over 10 parasites per thick film field) require urgent treatment. if you find a high parasite density.182 Manual of basic techniques for a health laboratory After procedure (i) or (ii). In this system. however. vivax may contain few parasites and therefore take more time to examine under the microscope. which would not be expected to have any effects on gametocytes. ovale and P. It is normal practice to count all the species present and to count and record separately the gametocytes of P. use clean slides and well-made and well-stained thick films.7. vivax). This is particularly important when monitoring the response to antimalarial drugs that are active against the schizont stage. Remember: For proper identification and reliable parasite counting. — the stage of development of the parasite. malariae or P. However. This system is less satisfactory. since they may reappear in the blood without reinfection.g. . Patients infected with P ovale or P vivax require additional treatment to eradicate . number of parasites counted number of leukocytes = number of parasites ml of blood Example: If 200 leukocytes are counted: (50 parasites ¥ 8000 (5 parasites ¥ 8000 200 leukocytes) = 2000 parasites ml of blood If 500 leukocytes are counted: 500 leukocytes) = 80 parasites ml of blood 2. If the result is negative. A simpler method of counting parasites in thick blood films is to use the plus system. falciparum and the asexual parasites. falciparum and P. falciparum and P. where it is known as sleeping sickness or African trypanosomiasis. and in Central and South America. specify: — the species of parasite found. report as “no parasites found”. state the result clearly in your report and send it immediately to the patient’s physician. use a simple mathematical formula. Trypanosomiasis is caused by infection with parasitic protozoa of the genus Trypanosoma. — the parasite density. it is necessary to differentiate the two species.3 Trypanosoma spp. A patient can harbour more than one species of malaria parasite at the same time (e. . Therefore. and should be used only when it is not possible to carry out the more acceptable count of parasites/ml of blood. the liver stages of these parasites.

b. sleeplessness and eventually severe headaches and blurred vision. From the site of the chancre. Between the first and second phases.e. sleeplessness.b. and months or years may elapse between the second and third phases. rhodesiense follows a more acute course and the phases are less marked. giving rise to occasional episodes of intermittent fever. pain in the joints and posterior lymph nodes of the neck. Heart complications are more frequent in trypanosomiasis caused by T. i. Sources of infection and modes of transmission African trypanosomiasis is transmitted by tsetse flies (Glossina spp. Invasion of the central nervous system causes irritability. Parasitology 183 African trypanosomiasis African trypanosomiasis occurs in three phases: — the acute phase — the parasitaemic phase — the neurological phase. 5 or 10 ml (both syringe and needle must be perfectly dry) Tincture of iodine q q q q q q . are easily seen under the microscope. rhodesiense In patients with African trypanosomiasis trypanosomes are found in the lymph glands in the early stage.) and humans are the main reservoir of infection. Pigs. 2–3 weeks after infection. rhodesiense. The most common symptoms of the first or acute phase are headache. Materials and reagents Microscope Microscope slides Coverslips Needle (for subcutaneous injection). the trypanosomes invade the bloodstream. psychotic phenomena. Transmission occurs when tsetse flies ingest the blood of infected humans or animals. Principle A drop of fluid from the lymph node is collected with a needle and examined immediately as a wet preparation.b. swelling of the eyelids and joints. Two or 3 days after the bite of an infected tsetse fly. which are motile flagellate protozoa. drowsiness. a chancre appears at the inoculation site. it disappears within 2–3 weeks.4. At a later stage the parasites may infect the central nervous system. and some patients die before reaching the neurological phase. The standard method of diagnosis of African trypanosomiasis in the early stage is to search for trypanosomes in aspirates from enlarged cervical lymph nodes. as well as epileptic attacks. 25-gauge Syringe. Trypanosomiasis caused by Trypanosoma brucei gambiense generally has a slow and chronic course. weeks or months can pass. but their role in spreading the disease is secondary. weight loss and generalized intense itching. The trypanosomes. Trypanosomiasis caused by T. paraesthesia. mental lethargy and coma. Examination of lymph node aspirates for Trypanosoma brucei gambiense and T. They disappear from the glands within 2–6 months. especially in the region of the breast bone. dogs and possibly other animal species can also harbour the parasite. It may cause death within a few months.

gently knead the gland. With the left hand. The glandular fluid will ooze into the needle. Collection of samples 1. then penetrate the centre of the gland. 4. Ask the patient to sit down. 4. With the right hand revolve the needle in both directions (Fig.141). 2. They do not become hard (except in chronic cases).141 Finding a lymph gland infected with trypanosomes q q 70% Ethanol Sodium chloride.142 Making the lymph gland stand out Fig. Hold your hand steady. Holding the needle between your thumb and forefinger. Fig. 4.143). 4. With the left hand. Affected glands are swollen and form a round lump 2–4 cm in diameter (Fig. The operation should take about 1 minute. 4. 4. They are elastic and slide under the skin.144). 0. from the base of the neck up to the ears.142). 5. Avoid the jugular vein and the arteries. 4. First pierce the skin.143 Introducing a needle into the lymph gland . introduce it at right angles into the centre of the gland (Fig. Feel both the right and the left sides of the neck. take the gland between the thumb and the index finger and make it stand out (Fig. 53).85% solution (reagent no. offering little resistance to pressure. Method Finding a lymph gland Lymph nodes are found among the cervical glands of the neck. Disinfect the chosen site on the neck with ethanol. 3.184 Manual of basic techniques for a health laboratory Fig. 4.

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Fig. 4.144 Twist the needle in both directions

Fig. 4.145 Collecting a sample of fluid from the lymph node

6. Attach the needle to the syringe, with the piston pulled back (Fig. 4.145). Push the piston gently halfway down the barrel to discharge a drop of the glandular fluid in the needle on to a slide. Microscopic examination Examine the slide first using the ¥ 10 objective, then change to the ¥ 40 objective to examine the parasites in greater detail. Close the condenser iris diaphragm sufficiently to give a sharp image. Wait until the convection currents stop. It is impossible to see the movement of trypanosomes among moving cells. Begin by examining the periphery of the preparation, near the edges of the coverslip, as shown in Fig. 4.146, as the trypanosomes tend to make their way to the edges. Then examine the rest of the preparation. The preparation will contain erythrocytes and leukocytes. Trypanosomes are about 20 mm long and are often hidden by cellular elements which are disturbed by the flagella of the trypanosomes as they move (Fig. 4.147). Examination of blood films for Trypanosoma brucei gambiense and T.b. rhodesiense Principle Trypanosomes belonging to the species Trypanosoma brucei are detected in the blood: — in wet preparations — in thick films after staining — following concentration by repeated centrifuging — in serological tests.
Fig. 4.147 Appearance of trypanosomes in a lymph node preparation

Fig. 4.146 Examining a lymph node sample under the microscope

Important: In African trypanosomiasis trypanosomes appear in the blood at intervals for a period of a few days, mainly during the first 3 months of the disease and especially during bouts of fever. Microscopic examination of capillary blood Materials and reagents q Microscope
q

Microscope slides

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Fig. 4.148 Collecting a capillary blood sample on each of two slides

Fig. 4.149 Collecting a capillary blood sample on filter-paper

q q q q q q q

Coverslips Blood lancets Filter-paper Sodium chloride, 0.85% solution (reagent no. 53) Giemsa stain (reagent no. 29) or Field stain (reagent no. 25) Buffered water, pH 7.2 (reagent no. 15) 70% Ethanol.

Method 1. Sterilize the pad of the third finger then prick with the blood lancet. Wipe away the first drop of blood with filter-paper. Collect two drops of blood (Fig. 4.148): — one drop on one slide — one drop on a second slide. 2. Collect two drops of blood on a piece of filter-paper (Fig. 4.149). Leave to dry. 3. On the first slide, place one drop of sodium chloride solution beside the drop of blood. Mix, using the corner of a slide (Fig. 4.150). Cover with a coverslip. 4. On the other slide, spread the blood to make a thick film (see page 174). Stain with Giemsa stain (see page 175) or Field stain (see page 177). Note: Blood films must be stained and examined immediately after collection of blood samples, since trypanosomes lyse and disappear within a few hours. Microscopic examination Wet preparation. Examine the first slide (with the wet preparation) under the microscope, using the ¥ 40 objective and reducing the condenser aperture. Examine the edges of the smear first. Look for movement among the erythrocytes; trypanosomes will displace them with their flagellum as they move forward. Make sure that the organisms are trypanosomes: Length: 15–25 mm (2–3 erythrocytes). Width: 3 mm (half an erythrocyte). Shape: like an elongated fish. Motility: trypanosomes move rapidly, advancing and contracting like a snake, and have an undulating membrane extending from a motile flagellum at the anterior end (Fig. 4.151).

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Fig. 4.150 Using a slide, mix the blood sample with saline solution

Fig. 4.151 Appearance of trypanosomes in a wet preparation

Do not confuse trypanosomes with microfilariae, which are much bigger (100–300 mm or 10–40 erythrocytes). Thick films. Thick films must always be examined, even if the examination of the wet preparation seems positive, to make sure that the motile organisms seen are trypanosomes. Trypanosomes of T.b. gambiense and T.b. rhodesiense are identical in appearance in stained preparations (Fig. 4.152): Length: 15–25 mm. Cytoplasm: pale blue. Nucleus: large central nucleus, stained reddish-purple. Granules: one compact red body at the posterior end: the kinetoplast. Undulating membrane: reddish-pink, starting at the kinetoplast. Flagellum: pink, extending 5 mm beyond the undulating membrane. If both slides are negative, repeat the tests for up to 7 days. Send the dried drops of blood on the strip of filter-paper to an immunological reference laboratory for testing for immunoglobulin M (IgM) and specific antibodies. Microscopic examination of venous blood concentrated by centrifugation Materials and reagents q Microscope
q q q q

Fig. 4.152 Appearance of trypanosomes in a stained thick blood film F: flagellum; K: kinetoplast; M: undulating membrane.

Microscope slides Coverslips Centrifuge or microhaematocrit centrifuge Conical centrifuge tubes with a mark at 10 ml or microhaematocrit capillary tubes Pasteur pipette Trisodium citrate, 3.2% solution (reagent no. 60).

q q

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Method 1. Pour 1 ml of trisodium citrate solution into a conical centrifuge tube. 2. Collect 9 ml of venous blood and add it to the trisodium citrate (i.e. fill the tube up to the 10-ml mark). 3. Mix and immediately centrifuge at 3000g for 3 minutes. 4. Draw off all the supernatant plasma and the layer of leukocytes above the deposit of erythrocytes. Expel this supernatant liquid into another tube (tube 2). Centrifuge at 3000g for 5 minutes. 5. Draw off all the supernatant fluid (but keep the deposit from tube 2). Expel the supernatant fluid into a third tube (tube 3). Centrifuge at 3000g for 10 minutes. 6. Examine the deposits of tubes 2 and 3 between a slide and a coverslip under the microscope. The trypanosomes will appear in the deposit from tube 3 (and occasionally in that from tube 2). Alternative method If a microhaematocrit centrifuge is available, venous (or capillary) blood with anticoagulant can be collected into a microhaematocrit capillary tube. The method of collection and examination is as for microfilariae (see page 164). Motile trypanosomes, if present, can be found in the plasma just above the layer of leukocytes. First use the ¥ 10 objective with reduced condenser aperture to detect any movement, then use the ¥ 40 objective to see the trypanosomes more clearly. Card agglutination trypanosomiasis test (CATT) for African trypanosomiasis The card agglutination trypanosomiasis test (CATT) is a serological test that is used for the diagnosis of African trypanosomiasis. Materials and reagents q Jar
q q q q q q q q q q q q q q

Tissue paper or cloth Glass stirring rods Dispensing vial droppers Rubber bulb for microhaematocrit tubes Syringes, 2.5 ml, with needles Blood lancets Heparinized microhaematocrit tubes Test cards Microhaematocrit tube holders with cover Manual or electric (12/220 V) rotator with cover Lyophilized antigen Lyophilized positive control serum Lyophilized negative control serum Buffer to reconstitute reagents.

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The above materials and reagents are available commercially as a test kit. The quantities supplied are sufficient to carry out 250 tests. Before carrying out the test, prepare your materials and reconstitute the amount of reagents required for the day’s work. Read and follow carefully the instructions provided in the kit. Method Collection of samples 1. Using a blood lancet, make a small puncture wound in the first, second or third finger of the patient. Collect the blood into a heparinized microhaematocrit tube (Fig. 4.153) until it is three-quarters full. 2. Immediately rotate the tube gently so that the blood runs from one end of the tube to the other (Fig. 4.154). Repeat the movement twice. This ensures that the blood and the heparin are mixed together, and prevents the sample from clotting in the tube.

Fig. 4.153 Collecting a blood sample using a microhaematocrit tube

Fig. 4.154 Rotating the tube to mix the sample

3. Place the microhaematocrit tube in the special holder (supplied with the kit; Fig. 4.155). Keep the holder covered as much as possible to avoid dust and to prevent the blood sample from drying in the tube. Microhaematocrit tube holders have 10 numbered slits. Make sure that you place the first tube in the first slit, etc. Once the holder is full, pass it to the person performing the test. Performing the test 1. Prepare two test cards. Place one drop of the reconstituted antigen in wells 1 and 2 of the first card and in all the wells of the second card. Hold the vial vertically to have constant calibrated drops (Fig. 4.156).

Fig. 4.155 A microhaematocrit tube holder

2. Using the first card, check the quality of the reagent. Place one drop of the reconstituted positive control in well 1 and one drop of the reconstituted negative control in well 2 (Fig. 4.157). Note: It is only necessary to do this once at the beginning of each day in a field survey.

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Fig. 4.156 Applying antigen to the test card

Fig. 4.157 Preparation of controls for the CATT

3. Using the second card, test the collected blood. Place one drop of blood from the first microhaematocrit tube in well 1, from the second tube in well 2, etc. (Fig. 4.158). Discard the microhaematocrit tube in a jar containing water with detergent. 4. Using a stirring rod, mix the reagents in each well of the first card and the reagents and blood samples in each well of the second card. Spread the mixture so that it covers the well (Fig. 4.159). Use a separate stirring rod for each well or clean the rod with a piece of tissue paper or a cloth between each well to avoid contamination of the samples. 5. Place both cards on the rotator, cover and set the timer to 5 minutes. If it is a manual rotator, check the time with your watch. The rotation speed should be slow, approximately 100g. If the rotation speed is too fast, clumps will settle at the edge of the wells; if it is too slow, the reaction will be weak. 6. After 5 minutes, examine the plates and record the reactions in each well. Do not allow the samples to dry out. If any samples have dried out, the test should be repeated.

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Fig. 4.158 Applying test samples to the test card

Fig. 4.159 Mixing the samples in each well of the test card

Fig. 4.160 Strong positive reaction in the CATT

Fig. 4.161 Weak positive reaction in the CATT

Results Strongly positive reactions (Fig. 4.160) Small or large clumps of particles are visible over the whole well or form a circle around the edge of the well. Weak positive reactions (Fig. 4.161) Very small clumps of particles are spread throughout the well or form a circle around the edge of the well. Repeat the test using serum or plasma.

162) No agglutination is visible. It can either persist indefinitely or may lead to the chronic form of the disease. a period of latent infection follows (indeterminate phase). The reaction remains uniform or occasionally slightly denser in the centre. but severe infections may be fatal. cutaneous lesions (chagomas) that resemble furuncles occur near the inoculation site. They will not keep and may give false results if used the next day. The infection of the digestive tract causes vomiting and diarrhoea. This type of reaction is usually negative. unless they have been refrigerated.192 Manual of basic techniques for a health laboratory who 1156 Fig.163 Nonspecific reaction in the CATT Negative reactions (Fig. African trypanosomiasis is also diagnosed in the laboratory by: — examining lymph node aspirates for trypanosomes (see page 183). Note: Discard any unused reconstituted reagents at the end of the day. page 259). On other areas of the face or body. Nonspecific reactions (Fig. About 50% of children manifest unilateral swelling of the eyelids (Romaña’s sign).3. The fever can be accompanied by myocarditis and meningitis. repeat the test using serum or plasma. The indeterminate phase is characterized by the presence of specific antibodies which can be detected by serological tests.162 Negative reaction in the CATT Fig. — inoculation of rats or mice with heparinized blood samples (only in specialized laboratories). — examining CSF specimens for trypanosomes (see section 8. — testing dried blood collected on filter-paper for IgM and specific antibodies (see page 187). If you are in any doubt about it.163) A dried-up ring is observed around the edge of the well or small dots or fine threads are seen. Patients suffering from the chronic form of the disease exhibit signs of cardiac insufficiency.3. but not by clinical symptoms. 4. Other diagnostic tests for African trypanosomiasis In addition to the tests described above. Chagas disease Chagas disease primarily affects children and is characterized by intermittent or continual high fever. 4. After the acute phase. 4. Primary infections can often pass unnoticed. Enlargement of the liver is common in children but not often seen in adults. There may be generalized oedema of the entire body. this phase is characterized by a low level of parasitaemia and absence of clinical symptoms. 4. Abnormalities in the electrocardiogram are often apparent although .

Parasitology 193 clinical symptoms are absent. Sources of infection and modes of transmission In Chagas disease the parasite (Trypanosoma cruzi) is transmitted by bugs of the genus Triatoma that become infected by ingesting the blood of infected humans or animals. cruzi.164) Length: about 15 mm in broad forms and 20 mm in slender forms. The same techniques are used as for African trypanosomiasis: — examination of a wet preparation (see page 186. Shape: broad forms are C-shaped. — examination of blood films prepared from centrifuged blood samples (see pages 187–188). rarely positive during the chronic stage of the disease). possibly because it passed asymptomatically or because it occurred in childhood and has been forgotten. 4. rangeli is not pathogenic. Patients with the chronic form of the disease may deny having ever experienced the acute form. and rarely thereafter.4. Although T. it must be identified and distinguished from T. Cytoplasm: pale blue. — examination of dried blood samples for IgM and specific antibodies (see page 187). During the chronic stage the diagnosis is based essentially on immunological methods. slender forms are generally S-shaped. The parasite multiplies in the intestine of the triatomine bug. 4. — examination of thick films (see page 187) repeated several days in succession. There is a serious risk that Chagas disease may be transmitted via blood transfusion if proper precautions are not taken. Important: Motile trypanosomes are found in the blood during the acute phase of the disease.164 Appearance of Trypanosoma cruzi in thick blood films . Humans are infected when the wound at the site of a triatomine bite is contaminated with the infected faeces of the bug. Fig. Diagnostic tests for Chagas disease Trypanosoma rangeli infects humans in almost the same areas as T. The trypanosomes that cause Chagas disease are difficult to find in the blood. cruzi for the diagnosis of Chagas disease. Identification of Trypanosoma cruzi in thick blood films (Fig.

humans are the main reservoir. Shape: only slender forms. Kinetoplast: small. it can be found both outside in the countryside and inside dwellings. The incubation period is generally from 2 to 6 months. multiply slowly in macrophages near the site of inoculation. near the posterior extremity. narrow. q q q Transmission can take place inside dwellings. the early phases of visceral leishmaniasis are characterized by chronic irregular fever. papules or nodules may appear in different parts of the body. chronic malaria and chronic leukaemia. wild animals and. Clinically.4 Leishmania spp. 4. but can vary from 10 days to several years. weight loss and — in some patients — patchy hypopigmentation of the skin occur. In Sudan. the lymph nodes. Flagellum: extending beyond the undulating membrane. cough. Nucleus: red. In India. It can affect the skin (cutaneous leishmaniasis). Flagellum: pink. liver and. In certain forms of cutaneous leishmaniasis plaques. where the amastigotes multiply rapidly. dogs are the main reservoir. Later. The vector feeds on dogs. In the Mediterranean basin and the Gulf area.194 Manual of basic techniques for a health laboratory Nucleus: large. like a dark red dot.7. Undulating membrane: visible. Undulating membrane: narrow. 4. which constitute microfoci of infection. Amastigotes of the Leishmania spp. mucous membranes (mucocutaneous leishmaniasis) and the reticuloendothelial system (visceral leishmaniasis or kala-azar). .165 Appearance of Trypanosoma rangeli in thick blood films 4. dark red or purple. which may be single or multiple. wild rodents and carnivores have been found to be reservoirs. Leishmaniasis is a group of diseases caused by infection with parasitic protozoa of the genus Leishmania. Some infected macrophages enter the bloodstream and reach the viscera. far away from the posterior extremity. Kinetoplast: large and round granule. The clinical symptoms of leishmaniasis may be similar to those found in schistosomiasis. occasionally. Fig. q In the Americas. extending beyond the undulating membrane.165) Length: 25–35 mm. with tapering extremities. diarrhoea and bleeding of the mucous membranes and secondary infections. progressive enlargement of the spleen. central and red. infection is spread to humans by the bite of the phlebotomine fly Lutzomyia longipalpis. and the vectors are various species of the genus Phlebotomus. Identification of Trypanosoma rangeli in thick blood films (Fig. Sources of infection and modes of transmission The epidemiology of the disease has unique features in each region and varies from one geographical area to another. less frequently. reddish-pink. humans. The disease occurs mainly in rural areas. In some patients a primary lesion forms several months before the other symptoms appear. Cutaneous leishmaniasis is characterized by skin ulcers. near the central part of the cell body.

29) Phosphate-buffered water.166). Tip off the methanol and flood the slide with the diluted Giemsa stain for 20 minutes. 2. Report the result as “amastigotes of Leishmania spp. may be found intracellularly in the macrophage cells or lying separately between the cells. turn the scalpel on to the flat side and gently scrape the base of the incision with the point of the blade. Rinse the slide in phosphate-buffered water and place it upside-down in a slide rack to drain and dry. Collect tissue cells. They measure 2–4 mm and have a prominent nucleus and a rod-shaped kinetoplast (Fig. dilute the Giemsa stain in phosphate-buffered water (1 volume of stain to 19 volumes of buffered water). 3.8 (reagent no. Allow the smear to air-dry and label the slide with a diamond pencil. Clean the edge of the ulcer using a swab soaked in ethanol. Slit skin specimens are collected from the edge of the ulcer. 2. present” or “not found”.4. . but avoid drawing blood. Both the nucleus and the kinetoplast stain red and the cytoplasm stains pale blue. The amastigotes of Leishmania spp. Fix the air-dried smears by flooding the slide with methanol for 2 minutes. pH 6. Method Collection of specimens 1. Typical leishmaniasis ulcers are cratered with a raised edge. Parasitology 195 Examination of slit skin smears for diagnosis of cutaneous leishmaniasis Principle Cutaneous leishmaniasis is diagnosed by demonstrating the typical amastigote stage of the organism from slit skin smears of ulcers. 4. Staining of smears 1.5 cm long and 2–3 mm deep. Still squeezing. 3. Materials and reagents q q q q q q q q q q Microscope Microscope slides Scalpel Gauze Slide rack Diamond pencil 70% Ethanol Methanol Giemsa stain (reagent no. 43). For use. Using the gauze pad compress the edge of the ulcer as firmly as possible to present a bloodless area. Microscopic examination Examine the slide under the microscope using the ¥ 100 oil-immersion objective. Use the scalpel to make a superficial incision along the edge of the ulcer about 0. Spread the material collected from the tip of the blade on to a slide in a circular motion to cover an area of 5–7 mm in diameter.

. usually after about 5 minutes.8) and in certain malignant diseases. Allow the tube to stand for 30 minutes. Collect 2–5 ml of blood into a centrifuge tube and allow it to clot. Results A positive result is shown by gelling of the serum — it becomes solid and turns white. Pipette 1 ml of clear serum into a test-tube. Note: Increased gamma globulin concentrations in serum are also seen following hepatitis B infection (see section 11. Separate the serum by centrifuging the tube for 3 minutes at 5000g or by leaving the tube overnight in a refrigerator or on the bench. 4. Add two or three drops of formalin to the serum.196 Manual of basic techniques for a health laboratory Formol gel test for visceral leishmaniasis This test is a non-specific indicator for the increased serum levels of gamma globulin that are seen in most patients with visceral leishmaniasis. amastigotes q q q Method 1. A negative result is recorded when there is no gelling or whitening of the serum. Materials and reagents q q Test-tubes Test-tube rack Centrifuge Centrifuge tubes Formalin (37% formaldehyde). 3. 2. such as multiple myeloma and Waldenström macroglobulinaemia.166 Leishmania spp. Fig. 4.

q q q q q 197 . q q The diagnosis of some diseases is also possible through serology. It may provide information of great value for the diagnosis. urine centrifugate. Make the loop with forceps. It is fixed with 70% methanol or by heating before being stained.1). The actual diameter of the loop should be 2 mm.3). The collection and dispatch of specimens to referral laboratories is.3.2 Materials and reagents q Inoculating loop: this is a metal wire (usually made of nickel–chromium alloy) fixed on to a handle and bent into a loop at the other end. therefore. direct microscopic examination of stained smears is an efficient way of studying the presence of bacteria in biological fluids that are normally sterile and in specimens from other sources. precise identification can only be obtained by culture. For example: q Specimens from male patients with urethritis at an early stage can be used to diagnose gonococcal infection with reasonable certainty (in females it is much more difficult). sputum. Microscopic examination of CSF is used in identifying the bacteria or fungi that cause meningitis (see section 8. Serological techniques are also important for epidemiological surveillance and early detection of diseases caused by bacteria that are difficult to culture (e.g.2.2 Preparation and fixation of smears 5. Mycobacterium tuberculosis). of utmost importance. 5. Bacteriology 5. Microscopic examination of sputum smears is a practical and effective technique for the detection of infectious cases of tuberculosis. Bacteriology 197 5. 5.) is prepared as follows: q q q The specimen is spread in a thin layer on a glass slide. Nevertheless. an example is syphilis (see section 11.1 Principle The sample to be examined (pus. etc. Microscope Microscope slides Coverslips Bunsen burner or spirit lamp 70% Methanol.1 Introduction Direct microscopic examination of smears is generally not sufficient to identify a bacterial species. 5. immediate treatment and control of the disease. CSF. It is allowed to dry completely.10).2. taking care that it is centred (Fig.5.

5). 5. 5.198 Manual of basic techniques for a health laboratory Fig. The side with the smear will not reflect the light. Allow it to cool (count to 20).1 Making an inoculating loop 5.3 Preparation of smears 1.4 Transferring a sample to a slide Fig.2).3). Unmarked smears are sometimes received in the laboratory from outside sources. 5. Let the slide dry completely in the air. then press the loop flat on to the centre of the slide (Fig. Repeat step 1.4). Flame the loop until it is red-hot: hold the loop just above the blue part of the flame. Still holding it flat against the slide. 5. 5.2 Flaming an inoculating loop Fig. To find out on which side of an unmarked slide the smear has been made. Take a portion of the specimen to be examined by placing the loop flat on the surface of the liquid (Fig. Leave a space between the specimen and each of the four sides of the slide. turn the slide so that it reflects the light from the window: q q The side without the smear will shine. Fig. 5.5 Preparing a smear . move the loop in an oval spiral.3 Collecting a sample using an inoculating loop Fig. 4. 5. as close to the vertical as possible (Fig.2. 5. Number a slide. 2. 3. 5. 5. outwards from the centre (Fig.

5.8 Gram staining reaction: fixation using iodine a: Gram-negative bacteria. neutral red or safranine solution a: Gram-negative bacteria. fix it by covering the slide with a few drops of 70% methanol for 2 minutes or by quickly passing the back of the slide through the flame three times (Fig. are not important. 5. The fixed smear can be stained as described in section 5.3 Staining techniques 5. 5.8).6).7). so that it can be seen more easily.2. b: Gram-positive bacteria. Vincent’s bacilli and Candida albicans.3. 5. Iodine solution fixes the violet colour more or less strongly in the bacteria (Fig. Bacteriology 199 5.1 Gram staining Fig. 5. 95% Ethanol: — decolorizes certain bacteria when the crystal violet is not strongly fixed by iodine solution (Fig. b: Gram-positive bacteria.9 Gram staining reaction: decolorization with ethanol a: Gram-negative bacteria.9 (b)). (b) (a) (b) q q (a) Fig. which remain dark violet (Fig. which are always present. Fig.4 Fixation of smears When the smear has dried completely. 5. 5. 5. b: Gram-positive bacteria. 5.7 Gram staining reaction: staining with crystal violet a: Gram-negative bacteria. (a) (a) (b) (b) Fig. Fig. It is sometimes useful to draw a circle around the smear with a grease pencil. Commensal bacteria. 5. — does not decolorize other bacteria when the crystal violet is strongly fixed by iodine solution (Fig.10 (b)).9 (a)).3. 5. 5.6 Fixing a smear over a flame Gram stain will enable the smear to be examined by microscopy for the presence of bacteria. Principle q q Crystal violet stains all bacteria deep violet (Fig. .10 (a)) — has no effect on the other bacteria. neutral red or safranine solution (pink): — re-stains (pink) the bacteria discoloured by ethanol (Fig.5. 5. pus cells.10 Gram staining reaction: restaining with carbol fuchsin. Carbol fuchsin. b: Gram-positive bacteria. They do not need to be considered for further examination or reported.

Cover the smear with carbol fuchsin for 2 minutes. Identification of specific organisms Candida albicans appears as large (2–4 mm in diameter) oval or round Grampositive spores (Fig 5. Microscopic examination First examine the slide using the ¥ 40 objective to see how the smear is distributed and then use the ¥ 100 oil-immersion objective. Fig. coliform bacilli. Cover the smear with crystal violet stain for 60 seconds. sometimes visible to the naked eye (white to yellow colour).14).g.1% solution (reagent no. 47).13(a)) with mycelium-like filaments of varying length with rounded ends (Fig. modified Hucker (reagent no. Decolorize rapidly with acetone–ethanol. etc. Wash off the stain with clean water and place the slide upright in a slide rack to drain and air-dry. 5. 16) (diluted 10fold with 95% ethanol). sputum. 5. enterococci. “Actinomycetes” are seen as large granules. 0. the periphery Grampositive (Fig. 40) or safranine solution (reagent no. 5. meningococci. Fix the smear as described in section 5. 3. 5. 0.4. salmonellae.2. staphylococci. anthrax bacilli). 2. The centre is Gram-negative.13(b)). pneumococci.1% solution (reagent no. Only 2–3 seconds are needed. 5. 6. Drain the slide and cover the smear with iodine for 60 seconds. 36) Acetone–ethanol decolorizer (reagent no. diphtheria bacilli.200 Manual of basic techniques for a health laboratory Materials and reagents q q q q q q Microscope Slide rack Crystal violet.12 Gram-negative organisms Gram-negative organisms Gram-negative organisms appear red (Fig. gonococci. 4) Carbol fuchsin solution for Ziehl–Neelsen stain (reagent no. shigellae. Method 1. cholera vibrios). 5.11 Gram-positive organisms Fig. 18) Lugol iodine. Fig. Gram-positive organisms Gram-positive organisms appear dark purple (Fig. streptococci.12) (e. 5. neutral red.g. micrococci. They are found in pus from skin.11) (e. 5. Wash off the stain with clean water. 4. Wash off the iodine with clean water.13 Candida albicans .

15 Vincent’s bacilli Vincent’s bacilli are seen as Gram-negative spirochaetes and fusiform rods (Fig. Sources of error A false Gram-positive reaction may occur because: q q q q q q The smear was fixed before it was dry. 7). Method 1. 5. The smear was too thick. The acetone–ethanol was not left on the slide long enough.14 “Actinomycetes” Fig. Fix the smear as described in section 5. A false Gram-negative reaction may occur because: q q The iodine solution was not left on the slide long enough.3.4. The acetone–ethanol was left on too long or not washed off properly. The iodine solution was not thoroughly washed off the slide.5. 5. Cover the smear with Albert stain for 3–5 minutes. . 5. Wash off the stain with clean water and place the slide upright in a slide rack to drain and air-dry. 5.16). Fig.2. No other bacteria should be reported as there are many commensal bacteria which may be confused with pathogens.16 Corynebacterium diphtheriae C. diphtheriae rods may be arranged in rows (a) or in V-formation (b) or joined at angles. 3. 5. This stain is used to show the dark-staining volutin granules that appear in Corynebacterium diphtheriae bacilli (see Fig. giving the appearance of Chinese characters (c). Materials and reagents q q q Microscope Slide rack Albert stain (reagent no. 5. 2. Bacteriology 201 Fig.15). The carbol fuchsin (or safranine or neutral red) solution was too strong or left on the slide too long. There was sediment in the bottle of crystal violet (filter before using).2 Staining with Albert stain (for the detection of Corynebacterium diphtheriae) If diphtheria is suspected a sputum smear should be stained with Albert stain.

Principle When mycobacteria and oocysts of Cryptosporidium spp. Tissues and other organisms are decolorized by the acid–ethanol solution and are demonstrated by a counterstain such as methylene blue. Mycobacterium leprae and oocysts of Cryptosporidium spp. If diphtheria is suspected. 16) (filtered before use) Table 5. they resist decolorization with a solution of acid or acid–ethanol and stain red. or joined at angles. The rods may be arranged in rows (a) or in V-formation (b). Corynebacterium diphtheriae appears as green rods (Fig. 5. Materials and reagents q q q q q Microscope Spirit lamp or Bunsen burner Slide rack Forceps Carbol fuchsin solution for Ziehl–Neelsen stain (reagent no.4. tuberculosis M. The presence of slender rods containing volutin granules is sufficient evidence for starting treatment for diphtheria. leprae M. bovis M. bovis Cryptosporidium spp. Mycobacterium spp.3. which stains them blue. giving the appearance of Chinese characters (c).16) containing green– black volutin granules. page 123). a specimen should be sent to the bacteriology laboratory for culture (see section 5.202 Manual of basic techniques for a health laboratory Microscopic examination First examine the slide using the ¥ 40 objective to see how the smear is distributed and then use the ¥ 100 oil-immersion objective. are stained with a hot strong solution of carbol fuchsin. 5.2.1 Organisms stained by Ziehl– Neelsen stain Sample Sputum Skin Urine Stool Gastric lavage Organism M. only resist decolorization with weak solutions of acid or acid–ethanol. They do not stain well with Gram stain or simple stains such as methylene blue.1). tuberculosis M. ulcerans M. (see section 4. bovis . and oocysts of Cryptosporidium spp. They are demonstrated using the modified Ziehl–Neelsen technique (Table 5. tuberculosis M. are referred to as “acidfast” due to their resistance to decolorization with acid solution. M.3 Staining with Ziehl–Neelsen stain (for the detection of acid-fast bacilli) Ziehl–Neelsen stain is used to identify mycobacteria and oocysts of Cryptosporidium spp.4).3.

6. 3. If red bacilli can be seen. Microscopic examination Examine the slide under the microscope. 5. Count the number of acid-fast bacilli present per microscope field (or per 100 microscope fields. 63). 39). diluted 1:1 with distilled water. Wash the slide well in clean water and cover the smear with malachite green or methylene blue for 1–2 minutes.5.2. report as “no acid-fast bacilli found”. If no acid-fast bacilli are seen. gently heat the slide over a spirit lamp or Bunsen burner until the stain starts to evaporate (at about 60 °C — do not overheat). report as “acid-fast bacilli present”. Wash off the stain with clean water and place the slide upright in a slide rack to drain and air-dry. 1% solution (see reagent no. .2 Reporting the number of acid-fast bacilli present Number of acid-fast bacilli present per microscope field < 0. 31). Using forceps. Then systematically examine the slide with the ¥ 100 oilimmersion objective to look for acid-fast bacilli (red bacilli). Method 1.1 (< 10 per 100 fields) 0. Materials and reagents q q q q Microscope Slide rack 70% Methanol Wayson stain (reagent no. 5.1–1 (10–100 per 100 fields) 1–10 > 10 Result Specify number present per 100 fields + ++ +++ q q Acid–ethanol for Ziehl–Neelsen stain (reagent no. first use the ¥ 40 objective to see how the smear is distributed. or methylene blue solution (reagent no.2. if very few acid-fast bacilli are present). 2.4 Staining with Wayson stain (for the detection of Yersinia pestis) Wayson stain is used to identify Yersinia pestis in bubo aspirates (see section 5. Leave the stain on the slide for 5 minutes. 4. Wash off the stain with clean water and cover the smear with acid–ethanol for 5 minutes or until the smear is pale pink. Bacteriology 203 Table 5. Fix the smear as described in section 5.10).4. Before moving to another slide.3. wipe the objective clean with lens tissue to prevent transfer of acid-fast bacilli to another slide. Examine the slide from end to end in steps until the whole smear is covered. Do not blot the smear. 5) Malachite green. Report the numbers of acid-fast bacilli present as described in Table 5. Cover the smear with filtered carbol fuchsin stain.

3. Materials and reagents q q q q Microscope Slide rack Potassium permanganate. the bacilli appear in chains (Fig. 5. q .11). 5. 5. 4% solution (reagent no.5 Staining with Loeffler methylene blue (for the detection of Bacillus anthracis) Loeffler methylene blue is used to stain Bacillus anthracis. Method 1. Fungi or yeasts: filaments of mycelium with or without pores. Microscopic examination First examine the slide using the ¥ 40 objective to check the distribution of the material and then use the ¥ 100 oil-immersion objective. The organisms include: q q Bacteria: Gram-positive and Gram-negative acid-fast bacilli.4 Examination of sputum specimens and throat swabs The presence of pathogenic organisms is revealed by microscopic examination of sputum specimens and throat swabs. Cover the slide with potassium permanganate for 10 minutes. 46) Loeffler methylene blue (reagent no. 3. Wash the slide in clean water and cover the smear with Loeffler methylene blue for 1 minute. They may be pathogenic or saprophytes that have multiplied in the sample after collection (correct identification by a specialized laboratory necessary). The staining procedure should be carried out in a safety cabinet. see page 200. Actinomycetes: granules. 2. 35). Gloves and protective clothing should therefore be worn when specimens suspected of being infected with anthrax are handled. 5. Wash the slide in clean water and place it upright in a slide rack to drain and airdry.17 Bacillus anthracis Microscopic examination First examine the slide using the ¥ 40 objective and then use the ¥ 100 oil-immersion objective. Wash off the stain with clean water and place the slide upright in a slide rack to drain and air-dry.17). Fig. 2. Fix the smear with methanol for 2 minutes. Bacillus anthracis appears as large blue rods surrounded by a mauve capsule. which causes anthrax (see section 5. Yersinia pestis appears as bipolar organisms which stain blue with pink ends.204 Manual of basic techniques for a health laboratory Method 1. 3. Cover the smear with Wayson stain for 15 seconds. Note: Anthrax is a highly contagious disease.

1. Secure the top and label the container with the name and number of the patient. very rarely. 5.5) Sterile cotton wool swabs Tongue depressor or spatula Test-tubes Sodium chloride crystals N-cetylpyridinium chloride Distilled water. 4. 6 cm long by 3 cm wide and as thin as possible. sterile cotton wool swabs should be prepared at a central-level laboratory. Plug with non-absorbent cotton wool.18). leakproof containers for sputum specimens.7. Prepare some thin sticks of wood (or aluminium wire). the following technique may be used. 5. Place in a glass test-tube.4.1 Materials and reagents q q q Microscope Microscope slides Wide-necked. otherwise.5.4). Prepare strips of cotton wool. 1. screw-topped jar containing 25 ml of the following solution: N-cetylpyridinium chloride Sodium chloride Distilled water to 5g 10 g 1000 ml.2 Method Collection of specimens Sputum specimens Sputum specimens should be collected early in the morning.5. Mould the swab into a conical shape. Culture is often necessary for the identification of the infective agents.5. 5. Fig. Check that a sufficient amount of sputum has been produced.1). If the specimen is to be dispatched to a laboratory for culture of Mycobacterium tuberculosis (see section 5. Sterilize (see section 3. spitting what he or she brings up into the container (Fig.5). 5. 2. ask the patient to expectorate directly into a wide-mouthed. q q q q q q If possible.4. 18 cm long and 2 mm in diameter. Important: Liquid frothy saliva and secretions from the nose and pharynx are not suitable for bacteriological examination. 2. 3.4. such as jars or stiff paper boxes (see section 2. Bacteriology 205 q Parasites: eggs of pulmonary flukes and. Ask the patient to take a deep breath and then cough deeply. .18 Collecting a sputum sample Screw on the top and label the jar with the patient’s name and the date of collection of the specimen (see section 3. eggs of schistosomes and adult worms of Mammomonogamus laryngeus. Ask the patient to produce another specimen. Roll the cotton wool round one end of the stick (or wire).

Report the numbers of acid-fast bacilli present as described in Table 5. 3. pus. report as “acid-fast bacilli present”.3. containing pus.2) and the other with Ziehl–Neelsen stain (see section 5. 2.3 Microscopic examination Examine the sputum with the naked eye and then by microscopy. Examine the smear stained with Ziehl–Neelsen stain as described in section 5. membraneous deposits or ulcers. 1 See also section 3. If no acid-fast bacilli are seen.3. If red bacilli can be seen. — mucoid: containing mostly mucus. Look carefully for signs of inflammation and any exudate. If there is blood present.3. . this must also be reported.3).2. Preparation of slides Prepare two smears from each of the specimens (see section 5. A sputum sample composed mostly of saliva will not be useful either for culture or for direct examination.4. Take care not to contaminate the swab with saliva. 5. Maximum preservation time: 10 days. The sputum of a person suffering from a bacterial infection usually contains: — thick mucus with air bubbles — threads of fibrin — patches of pus — occasional brownish streaks of blood.2.16) are seen.4.19). Examine the smear stained with Albert stain as described in section 5. report as “no acid-fast bacilli found”. After visual inspection report the appearance of the sputum as: — purulent: greenish. Use a sterile cotton wool swab to swab the infected area.4. — mucopurulent: greenish.2 and dispatched immediately to the laboratory. — mucosalivary: containing mucus with a small amount of saliva. If green rods containing green-black volutin granules (see Fig. Using a tongue depressor or a spatula to press the tongue down (Fig. containing both pus and mucus.4 Dispatch of specimens for culture1 Sputum specimens Sputum specimens are sent to a bacteriology laboratory for culture of Mycobacterium tuberculosis. antimicrobial susceptibility testing and inoculation into guinea-pigs. 5.7. 5. The specimen should be collected in a transport medium as described in section 5.19 Examining the back of the throat 5. examine the back of the throat. Fig.2. report as “Corynebacterium diphtheriae present”. Stain one smear with Albert stain (see section 5. 5.3.3).3.1. Return the swab to the sterile test-tube.206 Manual of basic techniques for a health laboratory Throat specimens 1.

Send the same day.21 Dispatching throat specimens in Stuart transport medium 5.5. .4. 5. Maximum preservation time: 3 days.20 Dispatching throat specimens in coagulated serum Fig. Fig. 5. If possible collect the specimen first thing in the morning before the patient passes urine. Rub the swab over the slanted surface of the serum. replace the swab in the sterile test-tube (see section 5.20).21).2) and send it immediately to the bacteriology laboratory.2 Method Collection of specimens From male patients 1. starting from the bottom (Fig. For confirmation of Corynebacterium diphtheriae infection If diphtheria is suspected. 56). Maximum preservation time: 24 hours. 5. Bacteriology 207 Throat specimens For routine investigation As soon as the specimen has been collected. If possible. Rub the swab over the surface of the medium from one side of the bottle to the other. 100 ml Pasteur pipette Cotton wool Amies transport medium (reagent no. the specimen should be sent in a sterile tube containing coagulated serum (which must be stored in a refrigerator).5. 5. starting from the bottom and not applying pressure (Fig. except for epidemiological surveys aimed at identifying carriers of meningococci.5 Examination of urogenital specimens for gonorrhoea 5. 5. 9). For detection of meningococci This is seldom necessary.1 Materials and reagents q q q q q q Microscope Microscope slides Bottle. use a transport medium such as Stuart transport medium. modified (reagent no.5. Send the same day.

22 Transferring a urogenital specimen to Amies transport medium 5. the specimen should be taken just before or just after the menstrual period. — no Gram-negative diplococci found.3 Microscopic examination Microscopic examination is of great value in the diagnosis of gonorrhoea in males: it is much less so in females.1).22). Preparation of slides Prepare a smear from each of the specimens. Gonococci Gonococci appear as Gram-negative diplococci (in pairs) (Fig. Extracellular Gram-negative diplococci may be seen if the pus cells are damaged. The nucleus may appear degenerated.3.5. Examine the slides using the ¥ 100 oil-immersion objective. From female patients The specimen should be taken from the cervical canal by a physician or specialist nurse. Use another swab to collect a drop of the pus for Gram staining (see section 5. In cases of chronic gonorrhoea. if no pus appears. . Culture is therefore necessary to isolate and identify the gonococci in specimens from females. 4. Clean around the urethral opening with sterile saline. Cocci appear oval and kidney-shaped. Collect a sample of the pus using a sterile cotton wool swab on a stick (see section 5. Apply gentle pressure on the penis so that a drop of pus appears on the meatus.23 Gonococci and pus cells a: Intracellular gonococci. Pus cells Pus cells have a pink nucleus and a colourless cytoplasm. Fig. 3. 5. where the elements are spread more thinly and are easier to see and the stain is less concentrated. 5. Insert the swab into a small bottle containing Amies transport medium. Cut the stick to allow the top to be tightened (Fig. 5.208 Manual of basic techniques for a health laboratory 2. 5. Report as: — Gram-negative intracellular diplococci present.4.23 (b)) should also be reported. Pay particular attention to the edges of the smears. 5. — Gram-negative extracellular diplococci present. Fig.23 (a)). 5. Extracellular Gram-negative diplococci (Fig.3.1). Leave the smears to air-dry and then stain with Gram stain (see section 5.1). gently massage the urethra from above downwards. A presumptive diagnosis of gonorrhoea can be made if Gram-negative intracellular diplococci are seen in smears from male patients. b: extracellular gonococci.

Round bottles may also be used. padded and plugged with cotton wool. rub the swab of pus over the whole surface of the solid medium. Other bacteria that cause infections in female patients All kinds of organisms are found in the smears.22).3. 5. Using a Pasteur pipette Method 1. diphtheria bacilli).g. Maximum preservation time: 6 hours (at ambient temperature).1. 3. 5. Bacteriology 209 Other bacteria that cause infections in male patients Numbers of the following may occasionally be seen in smears of urethral pus: — Gram-positive cocci (e. 2.1. Replace the cap on the bottle at once. The bottle should remain open for as short a time as possible to prevent the escape of gas. 56) is the best method.24. 5. 5. It is usually supplied in 30-ml flat bottles that contain 8 ml of solid medium (along one side of the bottle) and are filled with a mixture of air (90%) and carbon dioxide (10%). — Gram-negative cocci (saprophytes).g. 2.g.5.2. as shown in Fig. Collect the pus specimen on a sterile cotton wool swab as described in section 5. Collect the pus specimen on a swab as described in section 5. Unscrew the bottle cap. modified (reagent no. staphylococci).24 Dispatching urogenital specimens in a Pasteur pipette 1 See also section 3.5. from one side of the bottle to the other. if the medium can be obtained from a specialized laboratory.2. Holding the bottle as upright as possible (to prevent the gas escaping). particularly: — Gram-positive bacilli. — Gram-negative bacilli (e. .7.5. Method 1.5. coliform bacilli). Place the pipette in a sterile test-tube. This transport medium is also suitable for meningococci. Dispatch the bottle (at ambient temperature) immediately. Maximum preservation time: 3 days.6 Examination of genital specimens for syphilis Syphilis is a sexually transmitted disease caused by Treponema pallidum and occurs in three clinical stages.4 Dispatch of specimens for culture1 Using Stuart transport medium Sending the specimen in Stuart transport medium. — Gram-positive bacilli (e. Fig. but the shorter the delay the better. Place the bottle of medium upright. 3. Draw the pus specimen into a sterile Pasteur pipette plugged with cotton wool. These organisms are described in section 5. starting from the bottom (see Fig. 5.

It is characterized by granular papillomas on the skin. . collect a drop of the serous exudate and invert it immediately onto a slide. 1. The chancre area should be clear of any ointment before attempting to collect the specimens. Collect the chancre specimen with gauze moistened with sodium chloride solution. T. 53). 5.210 Manual of basic techniques for a health laboratory The primary stage is characterized by a painless genital ulcer (syphilitic chancre). Using a coverslip. 2.2 Method Collection of specimens Important: q q Wear protective gloves for this procedure. pallidum and T. pertenue are delicate. Yaws Yaws is caused by a non-venereal treponeme (Treponema pertenue) and occurs in humid tropical climates. Do not draw blood. gently scrape the edge of the ulcer with a sterile lancet or the flat edge of a scalpel blade (Fig. Secondary or tertiary syphilis may be transmitted to a fetus in utero (congenital syphilis). even when untreated.25 Collecting a chancre specimen 4.25). Compress the ulcer gently with a gauze pad. 5. tightly-coiled spirochaetes measuring 6– 12 mm ¥ 0. They are indistinguishable under the microscope.2 mm.6. 0. The chancre heals spontaneously. The tertiary stage is very rare and is characterized by central nervous system involvement and cardiac disease.1 Materials and reagents q q q q q q q Microscope with dark-field attachment Microscope slides Coverslips Gloves Gauze Sterile lancet or scalpel Sodium chloride. 5. 5.6. sometimes with enlargement of the lymph nodes in certain regions of the body. It is necessary to inspect samples suspected of being infected with spirochaetes by dark-field microscopy as they do not stain easily for viewing by transmitted light. In some patients the disease progresses to the secondary stage. Fig.85% solution (reagent no. 3. If there is no obvious serous fluid. The secondary stage results in: — skin rash — mouth ulcers — genital warts — generalized enlargement of lymph nodes.

With experience of dark-field microscopy.26 Treponemes 5.6.5. 5.26). This is done by assessing the functional characteristics of spermatozoa in the seminal fluid. Before collecting the semen specimen. 10 ml pH indicator paper Improved Neubauer counting chamber Sodium bicarbonate Phenol or formalin (37% formaldehyde) Distilled water Petroleum jelly.1 Materials and reagents q q q q q q q q q q q Microscope Microscope slides Coverslips Blood (Sahli) pipette Graduated cylinder. Bacteriology 211 5.3 Microscopic examination Examine the slide using the dark-field microscope. Fig. 5. prepare the semen diluting fluid as follows: — sodium bicarbonate — phenol or formalin — distilled water to 5g 1 ml 100 ml. the treponemes may be seen and can be distinguished from saprophytic treponemes by their size. 5.7 Examination of semen specimens Semen is investigated in patients to exclude infertility. characteristic movement and typical number of coils (Fig.7. .

9). Viscosity Freshly ejaculated semen should be completely liquefied within 30 minutes. preferably within 30 minutes.2–8.3. It cannot be examined immediately as semen is a highly viscous fluid and must “liquefy”.7. page 303–304). Less than 1. Then wash the slide gently with buffered distilled water. Colour Semen is normally an opaque grey colour. the tail takes up about 90% of the total length (Fig. let it dry in the air and then heat it very gently to fix. Stain the sperm with Leishman stain or Giemsa stain (see section 9. pH The pH is usually noted though it is of little significance. Fig.5 ml is considered abnormal.7. The normal volume is 4–5 ml.3 Macroscopic examination Volume Measure the volume in a small graduated cylinder — the amount varies from only a few drops up to 10 ml. with a large oval head. The head is 3–6 mm ¥ 2–3 mm.7. It does this within 15–30 minutes and should be examined as soon as possible after liquefaction has taken place. .10. After an extended period of abstinence from sexual activity it may appear slightly yellow. 5. a small neck and a long slender tail. Sperm MORPH system.4 Microscopic examination Normal spermatozoa are 50–70 mm in length.6 (range 7. Semen is always alkaline.212 Manual of basic techniques for a health laboratory 5.2 Method Collection of specimens The semen is collected by the patient in a clean.27 Normal spermatozoa Source: Image House Medical. Preparation of slides After liquefaction has taken place. Used with permission. Absence of liquefaction may interfere with sperm motility and fertilization. make a thin smear of semen on a slide (similar to a blood smear). dry bottle and is brought to the laboratory as soon as possible after collection. 5.27). 5. Remove the mucus (which will interfere with staining) by washing the slide with semen diluting fluid (see above). with an average pH of about 7. 5.

Used with permission.30 Spermatozoan with a double head Source: Image House Medical. Used with permission.28 Spermatozoan with an abnormally shaped head Source: Image House Medical.5. 5. Fig.29). — double heads (Fig. 5. 5. Bacteriology 213 Fig. Used with permission.29 Spermatozoan with an abnormally small head Source: Image House Medical. — abnormally sized heads (giant or minute) (Fig. Sperm MORPH system. Sperm MORPH system. The abnormalities of morphology to be looked for include: — abnormally shaped heads (Fig. 5.30). 5. . 5. Sperm MORPH system.28). Fig.

Sperm MORPH system. Used with permission.31). — epithelial cells. Fig. Sperm MORPH system. 5.31 Spermatozoan with a coiled tail Source: Image House Medical. — absent. their presence should also be noted. rudimentary or absent tails (Fig. 5. Normally about 80% of the spermatozoa are actively motile and about 20% are sluggish or not moving at all. In a normal smear there should not be more than 20% abnormal forms. Used with permission. Estimate roughly the proportion of motile to non-motile sperm forms in several different microscope fields. — polymorphonuclear leukocytes. Used with permission. also after 12 and 24 hours. draw semen to the 0. etc.33). 5. gently shake the specimen to mix. 5.5-ml mark. 5. Sperm count 1. After liquefaction has taken place. — immature cells from the testis. but after this there is an increasing loss of motility which at room temperature is often complete by 12 hours. Examine under the ¥ 40 objective of the microscope. Using a Sahli pipette. 2. During the examination of semen note the presence of any other cells such as: — erythrocytes. Observe the slide after 3 and 6 hours. For up to 3 hours there should be little or no reduction in motility. then draw in the semen diluting fluid to the 11-ml mark and place the pipette on a rotator to mix the contents.32 Spermatozoan with swollen neck Source: Image House Medical.33 Spermatozoan with double or rudimentary tails Source: Image House Medical. .32). 5. Sperm MORPH system. Motility Fig. — double. place a drop of semen on a slide.214 Manual of basic techniques for a health laboratory Fig. cover the drop with a coverslip and rim the edge with petroleum jelly to prevent evaporation. bifurcated or swollen necks (middle section) (Fig. — coiled tails (Fig. and if convenient. Decreased sperm motility may be a factor in infertility. Various crystals may also be seen. To check the motility.

The normal sperm count is between 60 million and 150 million per ml (100– 500 million/ml according to some sources).1 Materials and reagents q q q q Microscope Microscope slides Coverslips Sodium chloride. often with budding or short lengths of mycelium (see Fig.3 Microscopic examination Examine the Gram-stained slide using the ¥ 40 objective and the ¥ 100 oilimmersion objective. Preparation of slides 1.13). Use a microscope with the iris diaphragm closed to give good contrast. 2. Look for gonococci and Trichomonas vaginalis trophozoites in this preparation. 5. 53). respectively. so an additional multiplication factor of 1000 is needed. Transfer a small sample of discharge to a second slide. vulvovaginal candidiasis and trichomoniasis. with an undulating membrane and a prominent nucleus. Do not allow the specimen to dry out. 0. Examine the saline preparation as soon as possible using the ¥ 10 and the ¥ 40 objectives. Number of sperm ml = n ¥ 10 ¥ 20 ¥ 1000 4 where n = number of sperm counted. Trichomonas vaginalis trophozoites appear as highly motile flagellates measuring 8–20 mm. add a drop of saline solution and cover with a coverslip. though they may still be fertile. Candida albicans appears as large Gram-positive yeasts.12). except that the sperm count is per ml instead of per mm3.1) and examine for Candida albicans.3.8 Examination of vaginal discharge Vaginal discharge is examined by microscopy to exclude infections with gonococci. Make a smear of the discharge on a slide and allow it to air-dry. 5. Gonococci are Gram-negative and appear as small dots (see Fig. allow the sperm to settle and then count in the four corner squares.3). 5.8.5.8.85% solution (reagent no.2 Method Collection of specimens The specimen should be collected by a physician or specialist nurse. 5.8. 9. 5. which cause bacterial vaginosis. Bacteriology 215 3. Load an improved Neubauer counting chamber (see Fig. Patients with sperm counts below 60 million per ml definitely have low counts.6. The formula for calculation is similar to that used for leukocytes. . as for a leukocyte count (see section 9. 5. Stain the smear with Gram stain (see section 5. Candida albicans and Trichomonas vaginalis.40).

For infants or other patients who cannot produce a stool specimen.7. . 17) will preserve many kinds of enteric bacteria (cholera vibrios. 5. 2. in watery stool specimens. and all objects suspended in the saline solution appear bright.85% solution (reagent no. curved. 5. take a rectal swab.2 Method 1.9 Examination of watery stool specimens Dark-field microscopy is used to identify Vibrio cholerae and Campylobacter spp.).1. 53). 0. 3. Dip a sterile cotton wool swab in the stool specimen (Fig. 5. 1 See also section 3.3 Microscopic examination Use the ¥ 10 objective for focusing.9.1 Materials and reagents q q q q q Microscope with dark-field attachment Microscope slides Coverslips Inoculating loop Sodium chloride. etc.) for up to 4 weeks. 5. 5.216 Manual of basic techniques for a health laboratory 5. Place the slide on the microscope stage. Using Cary–Blair transport medium Cary–Blair transport medium (reagent no. shigella. other vibrios.2 g of stool in 5 ml of sodium chloride solution. Use the ¥ 40 objective to search for bacteria with characteristic shapes and motility (see below). Carefully remove any large particles. Moisten the swab with sodium chloride solution and introduce the swab into the rectum. etc. 1. Shigella. 2. Cover with a coverslip. are Gram-negative spiral rods that rotate rapidly on a central axis. Turn the swab several times with a circular movement (Fig. Open the iris diaphragm fully and place the dark-field attachment in position. Suspend 0. Vibrio cholerae appears as motile rods. 5. which may be short.9.35).9.9. 4. Allow the large particles to sediment. straight or involuted (Fig.34).4 Dispatch of specimens for culture1 It is often necessary to send stool specimens to a bacteriology laboratory for culture: — for the detection of cholera vibrios — for the detection of other bacteria that cause dysentery (species of Salmonella. salmonella. The uninoculated medium may be stored in a sealed bottle at room temperature for 8–12 weeks. 5.36). prepare a very thin smear on a slide. Campylobacter spp. Using an inoculating loop (sterilized by flaming). The background appears black.

discard it and prepare a fresh solution.34 Vibrio cholerae 3. 14) may be used. store it at room temperature. Bacteriology 217 Fig. Note: If the buffered glycerol saline has changed colour from pink to yellow. If you cannot send the swab immediately. Fig. Place the swab in a bottle containing Cary–Blair medium (three-quarters full) and send it to the bacteriology laboratory.35 Collecting a watery stool specimen Using buffered glycerol saline When specimens are to be sent for culture of enteric organisms other than cholera vibrios and Cary–Blair transport medium is not available. 5. Important: q q Never store the swab in the incubator.5. buffered glycerol saline (reagent no.36 Collecting a stool specimen from an infant . Never store the swab in the refrigerator. 5. Fig. 5.

where it causes localized swellings or buboes.3). Fill it to within 2 cm of the top with buffered glycerol saline.1 Materials and reagents q q q q q q q q Microscope Microscope slides Centrifuge Centrifuge tubes Specimen containers (see section 3. Report the appearance of the fluid. 5. 5.10 Examination of aspirates.10.3. This can only be done by an experienced physician as there is a risk of introducing infection.7) Inoculating loop 70% Methanol Reagents for: — Giemsa stain (see section 9. 2. A bijou bottle with a capacity of 7. exudates and effusions Aspirates. exudates and effusions are collected by inserting a sterile needle into the appropriate cavity.10. but can appear turbid or bloodstained.1) — Wayson stain (see section 5. Place the stool swab or rectal swab in the bottle and send it directly to the bacteriology laboratory. Cavity fluid is normally straw-coloured (yellow). which causes bubonic plague. Using an aseptic (sterile) technique. 5.5 ml is recommended.218 Manual of basic techniques for a health laboratory 1. Bubo aspirates are examined for Yersinia pestis. dry. transfer 10 ml of the fluid to a centrifuge tube and centrifuge at moderate speed (2000g) for several minutes.3. The organism is carried from the sites of inoculation to the lymph glands in the axillae.10. Preparation of slides Aspirated cavity fluid 1.3) — Gram stain (see section 5. sterile containers.2 Method Collection of specimens Aspirated cavity fluid Aspirated cavity fluid is collected into clean.4) — Ziehl–Neelsen stain (see section 5. .3. groin and neck. The cavities from which effusions can be collected include the following: — pleural (chest) — peritoneal (abdominal) — pericardial — synovial joint — bursa.

Bubo aspirates 1. 4. 5.2. 4% solution (reagent no.11 Examination of pus for Bacillus anthracis Bacillus anthracis is a pathogen of several types of animal. lymphocytes or mesothelial cells (from the lining of the cavity) and any atypical cells which may suggest cancer cells. 3.1) Loeffler methylene blue (reagent no. It is responsible for cutaneous anthrax where it shows in its early form as a blister on the skin often called a malignant pustule. Spread the fluid thinly over each slide (see section 5.10.2. Stain the slides with: — Gram stain (see section 5.3) — Giemsa stain (see section 9. Yersinia pestis is seen as bipolar organisms which stain blue with pink ends.3). 5. 35) Potassium permanganate. Bubo aspirates First examine the slide using the ¥ 40 objective to check the distribution of the material and then use the ¥ 100 oil-immersion objective to look for Yersinia pestis. send the slide to a bacteriology laboratory for culture. .3.4.3. 5.3.3 Microscopic examination Aspirated cavity fluid Examine each slide using the ¥ 40 objective and the ¥ 100 oil-immersion objective. resuspend the deposit and use an inoculating loop (sterilized by flaming) to prepare three smears.3). Fix the smear in methanol for 2 minutes and stain with Wayson stain (see section 5.11. Allow the smears to air-dry and fix with methanol. Remove the supernatant.10. If there are more than a few cells present or if the fluid is bloodstained. identify the predominant type of blood cell present — leukocytes.1 Materials and reagents q q q q q q q Protective clothing Gloves Microscope Microscope slides Inoculating loop or sterile cotton wool swabs (see section 5.4). 46).5. 2. Prepare a smear from the aspirated fluid as described in section 5.1) — Ziehl–Neelsen stain (see section 5. Look for acid-fast bacilli (mycobacteria) on the slide stained with Ziehl–Neelsen stain.3. Bacteriology 219 2. Look for any bacteria present on the slide stained with Gram stain. When examining the slide stained with Giemsa stain.

or curved clamp forceps with no teeth.1) Spirit lamp or Bunsen burner Reagents for Ziehl–Neelsen stain (see section 5.12.2.3) 95% Ethanol Sodium chloride.11.17). then stain with Loeffler methylene blue (see section 5.3. Prepare a smear from the pus or fluid as described in section 5.3.12 Examination of skin smears and nasal scrapings for Mycobacterium leprae Leprosy or Hansen disease is an infection of the peripheral nerve tissues by the bacterium Mycobacterium leprae.4.3 Microscopic examination First examine the smear using the ¥ 40 objective to check the distribution of the material and then use the ¥ 100 oil-immersion objective to look for Bacillus anthracis. Gloves and protective clothing must therefore be worn when specimens are collected.3. Leave the smear to air-dry in a safety cabinet. Diagnosis is made by examination of slit skin smears taken from various sites on the body. 5. 5. 53). Preparation of slides 1. 5. 2.5). or from nasal scrapings taken from the septum of the nose.11. q q q q q q q q . 5. the bacilli are arranged in chains (see Fig. which are chosen from areas where nerves run near to the skin surface.85% solution (reagent no. Fix the smear with potassium permanganate for 10 minutes. or tissue forceps Diamond pencil Gauze Small plastic sheets or gloves Sterile cotton wool swabs (see section 5. collect a few drops of pus or fluid from malignant pustules. These sites should include any nodules or patches on the face or the body.2 Method Collection of specimens Warning: Anthrax is highly contagious. Bacillus anthracis is seen as large blue rods surrounded by a mauve capsule. After fixation.1 Materials and reagents q q q q Microscope Microscope slides Scalpel Forceps with rounded ends and no teeth. Using an inoculating loop or a cotton wool swab. 0.220 Manual of basic techniques for a health laboratory 5. Slit skin smears are usually collected from six sites. smears are stained by the modified Ziehl–Neelsen method. The bacilli can be present in large numbers in lepromatous lesions (multibacillary leprosy) and are usually sparse or absent in tuberculoid lesions (paucibacillary leprosy).

Collect the serous tissue fluid and a small amount of cellular material.39). 5. Still squeezing with the forceps. if available. Squeeze the ear lobe or skin area hard using forceps (Fig. Specimens from the body and face 1. 5. these are pale or thickened. Flame the forceps and scalpel. but often larger (Fig.12.41).2 Method Collection of specimens Specimens from the ear and skin lesions 1. where the skin Fig. 5. This should be just inside the edge of the patch. look for any lesions or small swellings with a shiny surface (Fig.40). 5.37). use the edges of the ear lobe. If no lesion is visible. Examine the body and face for: — lesions similar to those found on the ear. flat patches (maculae) or plaques (Fig.38 Squeezing the ear lobe to stop the blood flow Fig. 2. infiltrated areas of skin which are similar in appearance to orange peel.39 Collecting a specimen from an ear lesion . or else use the forefinger and thumb to stop the flow of blood. 5. 4. Disinfect the area using a gauze swab moistened with ethanol.37 Leprosy lesions on the ear Fig.5. Choose the most acutely infiltrated lesion and select a site for collection of the specimen.38). 5. From each ear select the most congested lesion or nodule.5 cm long and 2–3 mm deep lengthwise in the middle of the lesion. From the skin lesion choose one area just inside the edge of a patch of depigmented area. Use the scalpel to make a superficial incision about 0. 5. — papules. but avoid drawing blood. Bacteriology 221 5. 5. turn the scalpel on to the flat side and gently scrape the base of the incision with the point of the blade (Fig. Examine the ear and skin in good light. 3.

(This is important.3. 5.222 Manual of basic techniques for a health laboratory appears to be altering most rapidly. Spread the serous material from the tip of the blade on to the slide in a circular motion until it covers an area of 5–7 mm in diameter (Fig. 4.3). Using the scalpel. Still squeezing with the forceps. Label the slide with a diamond pencil. Stain the smears using the modified Ziehl–Neelsen technique (see section 5. Leave the slide to dry in a dust-free place and then fix the smears by passing the back of the slide through the flame of a spirit lamp or Bunsen burner several times.40 Leprosy lesions on the arm Specimens are best prepared from an early morning “nose blow”. Between three and six specimens from the same patient can be placed on the same slide. 2. 5. to ensure that bacilli are detected. Stain the smears using the modified Ziehl–Neelsen technique (see section 5. Disinfect the incision with ethanol and apply a dressing if there is bleeding.3. 5. The patient blows his or her nose thoroughly into a small clean dry sheet of cellophane or plastic. Flame the clamp forceps and scalpel.42 Collecting a specimen from a skin lesion . scrape the bottom and edges of the incision with the tip of the scalpel. 2. 5. Specimens from the nose Fig.43). 5. Between two and four smears from the same patient may be prepared on a single slide.42).5 cm long and 2–3 mm deep with the tip of the scalpel (Fig.) A sample can also be taken from an area of skin showing the first signs of leprous infiltration.3). spread the specimen in a circular motion over an area 5– 7 mm in diameter on a glass slide labelled with a diamond pencil. Dry and fix the smears as for specimens from the ear or skin lesions (see above). 3. Preparation of slides Specimens from the ear and skin lesions 1. Fig. 2. Specimens from the body and face 1. 3. 3. Disinfect the area using a gauze swab soaked in ethanol. Collect a small amount of pulp and serous material. Squeeze the site hard using the forceps and make an incision about 0.41 Leprosy lesions on the face Fig.

Mycobacterium leprae are acid-fast bacilli. 5. 3. For example: . or — no acid-fast bacilli seen. straight or slightly curved with rounded ends. Recording the results Record the results as follows: — acid-fast bacilli present.5. The results of the examination can be graded as shown in Table 5. Note: Nasal smears sometimes contain non-pathogenic acid-fast bacilli that are not M. they may often appear granular with the rod being broken into several parts. Stain the slide by the modified Ziehl–Neelsen technique (see section 5. (b) in larger groups or clusters. leprae. Arrangement: The rods are arranged either in groups of 2–5 lying in parallel (Fig.44 (c)). leprae rods arranged: (a) in groups of 2–5 lying in parallel.44 (a) or in larger groups or clusters (Fig. Using a small cotton wool swab slightly moistened in sodium chloride solution. After staining by the modified Ziehl– Neelsen technique. fix the slide by passing the back of the slide quickly through the flame of a spirit lamp or Bunsen burner several times. 5. Bacteriology 223 Fig.44 Mycobacterium leprae M. Spread the material evenly on the slide and leave to dry. Shape: Largish rods.3 Microscopic examination Examine the slide using the ¥ 100 oil-immersion objective. and (c) in large numbers in circular masses (globi). Bacteriological index The bacteriological index (BI) is a guide to the bacterial load and is calculated by adding all the positive findings from all the body sites where a sample has been taken and dividing the total number of positive specimens by the number of sites. 5. they appear red on a blue background.12. occasionally large numbers in circular masses called “globi” can be seen (Fig. 2.43 Transferring the specimen to a slide Specimens from the nose 1. Size: 1–8 mm.3. 5. 5. 5. 4.3. When completely dry. Fig. transfer some of the nasal mucus from the plastic sheet to a labelled slide.44 (b)).3).

Count the numbers of bacilli that are uniformly stained red. Morphological index The morphological index provides an indicator of the viability of the bacilli.1–1 (1–10 per 10 fields) 1–10 10–100 100–1000 > 1000 Result 0 1+ 2+ 3+ 4+ 5+ 6+ — right ear — left ear — left arm — back 3+ 2+ 2+ 1+ The total number of positive specimens is 8 + and the BI is 8/4 = 2. the organism can be cultured in vivo in the foot pads of mice or in the armadillo. with no break in the rod. the morphological index is 8%. the number of viable bacilli is 8. However. These are considered as viable bacilli and if.3 Reporting the results of the examination for Mycobacterium leprae Number of bacilli per microscope field None (< 1 per 100 fields) 0. The morphological index is used for the initial diagnosis and follow-up of patients with multibacillary leprosy. for example. .224 Manual of basic techniques for a health laboratory Table 5.1 (1–10 per 100 fields) 0. It is determined as follows: Examine 100 bacilli on the prepared slide using the ¥100 objective.01–0. Culture There is no method available for the in vitro culture of Mycobacterium leprae.

Place a drop of lactophenol cotton blue mounting solution and a drop of 20% potassium hydroxide on to the scales and hair (Fig.2 Method2 Collection of specimens 1. 45). 1 2 A description of the method used for the identification of Candida albicans in vaginal discharge is provided in section 5. It can be found on the surface of the body. the scalp and the nails and between the toes. The hairs infected with tinea will appear fluorescent. 6. The circular lesions on the skin consist of a mass of branching hyphae. infected hair and nails may also contain spores of fungi. Note: Potassium hydroxide is a corrosive fluid and should not be allowed to touch the skin.1. Tinea infection can also be identified by examining the patient in a dark room illuminated with ultraviolet light. The strong alkali will dissolve the keratin in the tissue. 20% solution (reagent no. 3.6. Cover with a coverslip.8. 2. 6.1. 225 . Clean the infected area with a cotton wool swab soaked in ethanol.1 Examination of skin and hair for fungi Ringworm or tinea is a fungal infection of the skin. 33) Potassium hydroxide. enabling hyphae and spores to be seen. Cross-infection between humans frequently occurs and infection can also be acquired from infected animals or soil. Use a sterile scalpel to gently scrape the edge of a lesion and collect some skin scales on to a glass slide or on to a piece of dark paper on which the scales can be more easily seen. Also collect a few broken or damaged hairs from the infected areas of the scalp using broad tweezers and place them on the slide.1). Mycology1 6.1 Materials and reagents q q q q q q q q q q q q Microscope Microscope slides (or dark paper) Coverslips Scalpel Tweezers Petri dish Bunsen burner or spirit lamp Cotton wool swab Cotton wool 70% Ethanol Lactophenol cotton blue mounting solution (reagent no. 6.

Microscopic examination Examine the cleared specimen using the ¥10 and ¥40 objectives.3 Ectothrix a: Immature spores. where it is called “Madura foot”. 6. Mycetoma produce small granules which are discharged through sinuses to the surface. These spores are known as ectothrix.3). Fungal hyphae can be differentiated from other tissue structures by their branching and cross walls or septa. Spores (large round granules with transparent membranes) may be seen around the outside of the hairs (Fig. b: mature spores. Branching hyphae and chains of angular rounded arthrospores may be seen. .4). clear the specimen by holding the slide above a Bunsen burner or spirit lamp flame for 1 minute (Fig. depending on the thickness. 6. Report as “fungal hyphae or spores present” or “not found”.4 Endothrix a: Immature spores. Place the slide in a covered Petri dish with some damp cotton wool to prevent the specimen drying out. b: mature spores. Adjust the iris diaphragm of the condenser to give good contrast. The most commonly infected site is the foot. 6.2 Clearing the specimen above a flame Fig.1 Preparation of slides for microscopic examination of skin and hair for fungi Fig.2 Examination of pus for mycetoma Mycetoma is a chronic granulomatous disease of the subcutaneous and deep tissue.2). Fig. 6. They stain blue with lactophenol cotton blue.226 Manual of basic techniques for a health laboratory Fig. 6. 4. Alternatively. 6. Leave the specimen to clear for 5–30 minutes. Other possible sites of infection include the hands. 6. the head and the chest wall. These granules are used in the diagnosis of the disease. Spores found inside the hairs are called endothrix (Fig. 6.

which appear: — pale and discoloured in dark-skinned patients. 45) Reagents for Gram staining (see section 5. Crush a few granules in some distilled water and place on two slides. 53) Potassium hydroxide.2. 5.3 Examination of skin for pityriasis versicolor Pityriasis versicolor is a common skin disease in hot climates.1 Materials and reagents q q q q Microscope Microscope slides Adhesive cellophane tape Tongue depressor or glass rod . 4. Microscopic examination Examine the cleared specimen using the ¥ 10 and ¥ 40 objectives. 20% solution (reagent no.3. Add a drop of saline or water.3.1 Materials and reagents q q q q q q q q q Microscope Microscope slides Coverslips Sterile needles Distilled water 70% Methanol Sodium chloride. it is caused by the fungus Pityrosporum furfur. 6. Look for branching and twisted hyphae or fragmented threads.3.85% solution (reagent no. The face and body are covered with patches. “Gram staining shows Gram-positive thin hyphae” or “Gram staining does not show Gram-positive thin hyphae”. Gram-stained granules may show thin or fragmented Gram-positive threads. fix with methanol for 2–3 minutes and stain with Gram stain (see section 5. 6. 0. size. 2. spread the pus gently and look for granules. 3. 6. Use a sterile needle to lift the surface crust over a sinus.1). — yellowish-brown in white-skinned patients. Allow one slide to air-dry.2. 6. Mycology 227 6. Leave the specimen to clear for 10 minutes. Granules vary in colour.1). Report as: q q “pus from sinus containing granules [specify colour and size] present”. Carefully remove some of the discharging pus on to a slide. Adjust the iris diaphragm of the condenser to give good contrast. Place a few drops of 20% potassium hydroxide on to the second slide and cover with a coverslip. shape and degree of hardness.6.2 Method Collection of specimens 1.

Place it over the patch so that it overlaps one edge (Fig. Moisten it with a gauze pad dipped in the eosin solution (Fig.5). 6.7 Collecting a skin specimen .3. Cut a strip of adhesive tape about 5 cm long.7). 6. Choose a rapidly developing patch of infected skin. 6.5 Staining pityriasis versicolor-infected skin patches with eosin Fig. 6. 1% solution (reagent no. Stick the tape on the skin and press firmly from one end to the other. Leave to dry for 1 minute. (Do not take the specimen if talcum powder has been used on the skin. 6. if possible (otherwise.2 Method Collection of specimens 1. Place it immediately on a microscope slide. Pull the adhesive tape away with forceps. sticky side down (Fig. 23). 6. passing a tongue depressor or glass rod over it several times (Fig. Wash it off first. Fig. 6.8). 3. examine without staining).228 Manual of basic techniques for a health laboratory q q q Forceps Gauze pads Eosin.6 Applying adhesive tape to a skin patch Fig. 6.6).) 2.

9).10 Pityrosporum furfur (¥ 100) ¥ . with branches. Budding is sometimes visible. bent and twisted rods. 6. The spores appear white on a pink background if the skin was treated with eosin. and are also visible in unstained preparations. 6.8 Transferring a skin specimen to a slide Fig. Spores Size: 3–8 mm diameter. 6. 6.10). thick-walled. arranged in a bunch or cluster. Fig. Shape: finger-shaped. Mycology 229 Fig. Shape: round or slightly rectangular. Change to the ¥ 100 oil-immersion objective to examine the details (Fig. 6.6. Mycelium filaments Size: 20–40 mm long and 5 mm wide.9 Pityrosporum furfur (¥ 40) ¥ Microscopic examination Examine the whole slide under the microscope using the ¥ 40 objective until a cluster of large granules (the spores) is seen (Fig.

.

7. Examination of urine 231 Part III .

232 Manual of basic techniques for a health laboratory .

Usually. All the urine passed during the rest of the day and night is collected in the bottle. a specimen collected in the normal way following thorough cleansing is acceptable for this purpose. The procedure is used for certain bacteriological tests. There are also many patients who exhibit no clinical symptoms. 24-Hour urine specimen The 24-hour urine specimen is collected in a clear 2-litre bottle with a stopper. 233 . mainly in women. Measure the volume of urine with a measuring cylinder and record it. Random urine specimen A random urine sample.1 Types of urine specimen Early morning urine specimen Early morning urine provides the most concentrated sample. the patient places an open container in the stream of urine and collects about 20 ml of urine. 7. clean and dry. The next morning the patient gets up and collects the first urine of the morning in the bottle. Examination of urine 233 7. however. but in whom previously unrecognized urinary tract infections can be diagnosed by urine examination. will enable the laboratory to screen for substances which are indicators of kidney infection. this urine is not collected. Examination of urine Examination of urine is a fundamental investigation in patients in whom kidney disorders or infections of the urinary tract are suspected. Midstream urine specimen While passing urine. On the first morning the patient gets up and urinates. taken at any time of the day. 7. Urine specimens collected using a catheter Collection of urine using a catheter must be carried out by a qualified physician or nurse.7. If the urine specimen has to be transported for any length of time it should contain an appropriate preservative to prevent bacterial overgrowth or hatching of viable ova. The bottle should then be taken immediately to the laboratory. The container should be covered immediately. Terminal urine specimen The patient urinates the last portion of urine into an open container.1.1 Collection of urine specimens Containers for the collection of urine should be wide-mouthed.

If urine has been collected to check for the presence of Schistosoma haematobium ova but it may not be examined for several hours.g.2 Testing for the presence of blood Elevated erythrocyte counts and haemoglobin levels may occur in urine: — after heavy physical exercise. They are then compared with a comparison chart after an appropriate time that is also specified on the chart. Dipsticks must be stored according to the manufacturer’s instructions. Urine can occasionally appear colourless.7). schistosomiasis).2. — in acute cystitis or urethritis. . pale yellow. — in acute glomerulonephritis. Method The dipsticks are placed into the urine and immediately removed.1. — colourless. The colour changes observed on the dipstick will give a semi-quantitative estimation of the amount of substance present.234 Manual of basic techniques for a health laboratory Urine specimens from infants Urine can be collected into a plastic bag with an adhesive mouth. 2). This can be reported as negative.g. 7.2. nitrite and leukocyte esterase). +++. The bag is fixed around the genitalia and left in place for 1–3 hours. Pigments from bile substances may make the urine appear deep yellow or brown. blood. The presence of blood cells or excess salts may make the urine appear cloudy. deep yellow or brown. More concentrated urine may appear dark yellow. Blood cells are easily seen by microscopic examination after centrifugation (see section 7. 7. Lysed erythrocytes can be detected using a urine dipstick which has a segment for detection of blood. q 7. Urine dipsticks are available for detection of a single substance (e.1 Appearance q Urine is normally clear straw-yellow in colour.2 Preservation of urine specimens q Urine passed at a clinic and examined immediately does not require preservation. glucose or protein) or for detection of several substances (e. q q q Report the appearance as: — clear or cloudy. — in vaginal tract infections.2. depending on the examination requested. +. ++++ or as an approximate value of the concentration of the substance tested for. ++. — in patients suffering from certain tumours.2 Examination of urine specimens 7. — in parasitic infections (e. it should be acidified with a few drops of 10% acetic acid (reagent no. Colostomy bags can be used.g.

0. Let a few drops of fresh urine fall from the dropper on to the paper (Fig. Fig.1 Materials for measuring the pH of urine q q Materials (Fig. Repeat the test in another watch glass.g.0) are common in patients with infections of the urinary tract and in people on a vegetarian diet. 7.4 Checking the pH using indicator paper of limited pH range .2 Applying the urine specimen to universal indicator paper q The urine specimen must be tested within 1 hour of collection. use indicator paper for the range 5. using the paper for the corresponding limited range.0).0–7.0–8. pH = 6. 7. Examination of urine 235 7.0. use indicator paper for the range 6. select a strip of indicator paper for the corresponding limited range.0–7. According to the result obtained. 7.7. Compare the colour obtained with those shown on the standard chart (Fig. Principle q Coloured indicator paper is dipped in the urine (or placed in a watch glass and a few drops of urine are added to it). 3. 7. In certain diseases the pH of the urine may increase or decrease. dip the test paper directly into the urine in the receptacle. If the pH is 7 or more. Read off the pH unit given for the colour that matches the test paper most closely.0.0 range and for the 6.5–5.3 Measuring the pH Normal freshly passed urine is slightly acid. Alkaline pH values (7. 7. with a pH of around 6.0 range.2.1) q q q q Watch glasses Dropper Forceps Universal indicator paper (for measuring pH from 1 to 10) Indicator paper of limited pH range: for the 5. 4.0 (range 5. Read off the pH of the urine on the standard chart (Fig.3 Checking the pH using universal indicator paper Fig.8– 8.3). e. Acid pH values (4. Fig. For example.5) are observed in some forms of diabetes.0–7. Pick the strip of paper up with forceps. if the pH is 6. Alternatively. 7. Fig. Place a strip of universal indicator paper in a watch glass.5.4). The paper is then compared with a standard control chart giving the corresponding pH value. The pH of urine is normally about 6. 7. Method 1. 2.2 or pH = 7. muscular fatigue and acidosis. The colour changes according to the pH.0–8.2). 7.

carbonates. Add eight drops of urine and mix well.4 Detection of glucose Principle Glucose is the most commonly found sugar substance in urine. urates. pages 245–248). crystalline deposits in urine have no diagnostic significance. Glucose is a reducing substance. 10). uric acid.2. It reduces the blue copper sulfate in Benedict solution to red copper oxide.1. Boil over a Bunsen burner or spirit lamp for 2 minutes (Fig. Lactose is also a reducing sugar and is occasionally seen in the urine of pregnant women.5). 2.7. others only in alkaline urine. Materials and reagents q q q q q q q q Test-tubes Wooden test-tube holder Test-tube rack Beaker or can Bunsen burner or spirit lamp Dropper pipette Graduated pipette.236 Manual of basic techniques for a health laboratory pH and crystalline deposits Determination of the pH of urine is useful for the identification of crystalline deposits (see section 7. 7. Method 1. 3. particularly in diabetic patients and patients suffering from chronic renal failure. Place the test-tube in the test-tube rack and allow to cool to room temperature. 7. 7.2. or place the test-tube in a beaker or can of boiling water for 5 minutes. Some crystals are deposited only in acid urine.5 Benedict method for detection of reducing substances in urine Glucose in urine can also be detected using a urine dipstick (see section 7.2).2. 7. For example: — acid urine: oxalates.2. which is insoluble. — alkaline urine: phosphates. Examine the colour change of the solution and any precipitate. 4. Fig. Except in very rare diseases. Report the result as shown in Table 7.5 Detection and estimation of protein Elevated protein levels are observed in the urine of patients with: — urinary schistosomiasis — chronic renal disease . Pipette 5 ml of Benedict solution into a test-tube. 5 ml Benedict solution (reagent no.

62). ed.005% solution (prepared from albumin stock standard. If albumin stock standard is not available. has no pathological significance. American journal of clinical pathology. a precipitate is formed.1 Reporting the results of the Benedict method for detection of reducing substances in urine Colour Blue Green Green with yellow precipitate Yellow/dark green Brown Orange to brick red Result Negative Borderline + ++ +++ ++++ — pyelonephritis — diabetes mellitus — systemic disorders (lupus erythematosus) — multiple myeloma. Turbidimetric measurement of total urinary proteins: a revised method. 2nd ed. including albumin and globulins. 81:651–654. WB Saunders. orthostatic proteinuria. the working standard can also be divided into aliquots and stored at –20°C for up to 6 months. Ash KO. 1994. . which occurs on standing up and disappears on lying down. diluted 1:100 with sodium chloride. a form of functional proteinuria usually seen in young men. As with the albumin standard. Textbook of clinical chemistry.7.0% solution. q q q The albumin working standard can be divided into aliquots and stored at -20°C for up to 6 months. 5. 0. Examination of urine 237 Table 7. However. 53) Positive and negative controls Albumin working standard. which is measured by turbidimetry. 1984. Brown PI. 0. Principle1 When trichloroacetic acid is added to urine containing protein. commercial serum-based standards containing both albumin and globulin can be used to prepare a working standard solution of the appropriate concentration. 53)). Materials and reagents q q q q q q q Spectrophotometer Test-tubes Test-tube rack Centrifuge Mechanical rotator Bovine or human serum albumin Trichloroacetic acid.85% solution (reagent no. 0.85% solution (reagent no. 5% solution (see reagent no. diluted 1: 4 with distilled water Sodium chloride. This reaction occurs with almost all proteins. 1 Further details of the method described here are given in the following references: Shahangian S. Tietz NW. Philadelphia.

measure and record the optical density of the tests and blanks at 620 nm. Leave to stand at room temperature for 10 minutes.238 Manual of basic techniques for a health laboratory Method Collection of specimens Random. If this method is used to screen for microproteinuria (which may be correlated with microalbuminuria in the absence of tubular damage. Calculation Calculate the concentration of protein in the urine specimen using the following formula: OD T . however. the specimens should be stored at -20°C. Specimens that are collected over 24 hours should be stored at 4–8°C during the period of collection.OD TB ¥ C ODR . any positive results should always be confirmed by repeating the test on one or more separate samples. measure and record the optical density of the tests and blanks at 405 nm. The analytical range of measurement using this method is 100–1000 mg/l. 2. 4. No preservatives should be added to the specimens. The spectrophotometer should be set to zero with distilled water before any measurements are taken. 3. as described below.1). Technique 1. q q . Centrifuge the test blanks at 2000g for 10 minutes. timed or 24-hour urine specimens should be used (see section 7. Add 0.1. It should also be calibrated. Collected specimens should be kept at 4°C until analysis. Leave all the tubes to stand at room temperature for 35 minutes after mixing. Note: q If a serum-based control is used for calibration purposes. Using the spectrophotometer. Because the amount of protein excreted in the urine may vary greatly. an independent material must be used for the purpose of quality control.4 ml of trichloroacetic acid solution to all of the test-tubes and mix well. urinary infections and treatment with certain drugs) in high-risk populations such as patients with diabetes. Repeat the process with the working standard and the control. to prevent bacterial growth. Add 1. If analysis is likely to be delayed for more than 24 hours. Using the spectrophotometer. 4.6 ml of the urine specimen to each of two test-tubes (test and test blank). the following modifications should be applied to steps 2 and 4: 2.ODRB where: C = concentration of the calibration solution ODR = optical density of the working standard ODRT = optical density of the working standard test blank ODT = optical density of the test specimen ODTB = optical density of the test specimen blank.

vomiting.2). Method 1. Protein in urine can also be detected using a protein dipstick (see section 7. 2. 7. 7. 5.6 Preparation of sodium nitroprusside solution Fig.7).2. Fig.6). Holding the tip of the pipette against the side of the tube.7. let 20 drops (1 ml) of ammonia solution flow on to the surface of the liquid (Fig.6 Detection of ketone bodies Normal urine does not contain ketone bodies. 4. Shake well until the crystals are almost dissolved. Add four drops of acetic acid to the urine. Principle When sodium nitroprusside (sodium nitrosyl pentacyanoferrate (III)) is added to urine containing ketone bodies.) 3. Add 5 ml of distilled water. 7. Just before carrying out the test. place a sufficient number of sodium nitroprusside crystals into a test-tube to cover the bottom (Fig. Measure 10 ml of urine into another test-tube. Wait for 5 minutes before reading — a positive result may be obvious before this time. followed by 10 drops of freshly prepared sodium nitroprusside solution. 10 ml Dropping pipette Sodium nitroprusside crystals Acetic acid Ammonia.7 Adding ammonia solution to the surface of the sodium nitroprusside solution . a purple colour is produced. Acetone and other ketone bodies may appear in urine: — in severe or untreated diabetes. prolonged starvation and following strenuous exercise). (Not all the crystals are expected to dissolve as the solution is saturated. malnutrition. 7. 7. Materials and reagents q q q q q q q Test-tubes Test-tube rack Measuring cylinder. Mix well. Examination of urine 239 The analytical range of this modified method is 25–700 mg/l.2. — in certain other conditions (dehydration.

7 Detection of abnormal elements Fig.8). 15 ml Pasteur pipette Coverslips Formaldehyde Distilled water. The following abnormal elements may be found: — leukocytes — abnormal numbers of erythrocytes — abnormal crystals (very rarely) — parasitic trophozoites (e.2. Schistosoma haematobium. 7. 7. In certain diseases of the urinary tract. 1 See also section 7.2).2. no colour change occurs. a purple ring appears on top of the urine. b: negative reaction.g. Principle Urine contains cells and crystals in suspension that can be collected by centrifugation or by allowing the urine to stand and the suspended particles to form a sediment. .2. Ketone bodies in urine can also be detected using a urine dipstick (see section 7.8. Trichomonas vaginalis) or ova (e.2.8 Test for ketone substances in urine a: Positive reaction. the urinary deposits are considerably altered.g. If the result is negative.1 Enterobius vermicularis) — bacteria — fungi — abnormal casts. Report the result as shown in Table 7.2 Reporting the results of the test for detection of ketone bodies in urine Colour change None Pink ring Red ring Purple ring Result Negative + ++ +++ If the result is positive (Fig. Materials and reagents q q q q q q q q Microscope Microscope slides Centrifuge Conical centrifuge tube.240 Manual of basic techniques for a health laboratory Table 7. 7. The resulting urinary deposit can be examined under the microscope.

scan the coverslip all over to look for ova of Schistosoma haematobium when indicated. (The supernatant may be used for biochemical testing. 2. Erythrocytes (Fig. The specimen can be preserved for microscopic examination of the deposit by adding 8–10 drops of formaldehyde. .9) Erythrocytes in urine may be: (a) intact: small yellowish discs. 3. Resuspend the deposit in distilled water and mix by shaking the tube. Pour off the supernatant by quickly inverting the tube without shaking. 6. 5. (b) crenated: spiky edges. 7. b: crenated cells.1. The following may be found in urine: — erythrocytes — leukocytes — epithelial cells — casts — fungi — crystals — parasite eggs and larvae — Trichomonas vaginalis — spermatozoa. A midstream urine specimen (see section 7. Fig. reduced diameter (5–6 mm). darker at the edges (8 mm). Examination of urine 241 Method Collection of specimens Urine to be examined under the microscope must be freshly passed into a clean dry vessel. 7. 10% solution (reagent no. (c) swollen: thin circles. Using the ¥ 40 objective and with the condenser lowered or aperture reduced. Label the slide with the patient’s name or identification number.7. Centrifuge the specimen at medium speed (2000g) for 5 minutes.1) is the most useful. There are normally very few erythrocytes in urine. The shape of the cells often changes during storage of urine and does not have any diagnostic importance. scan the coverslip area again and report any findings as a quantitative value for each high-power field. Microscopic examination Using the ¥ 10 objective and with the condenser lowered. Urine preserved in this way is not suitable for other tests. 28) per 300 ml of urine. increased diameter (9–10 mm).9 Erythrocytes a: Intact cells. Mix the urine specimen gently and pour approximately 11 ml into a centrifuge tube.) 4. Preparation of the deposit 1. Transfer one drop of the deposit on to a slide using a Pasteur pipette and cover with a coverslip. Urine stored in a refrigerator may contain an excess of precipitated salts and will not be suitable for microscopy. c: swollen cells.

7. 7. 7. indicates a urinary tract infection. (b) degenerated: distorted shape.10) Leukocytes found in urine may be: (a) intact: clear granular discs. Using the ¥ 40 objective. How to express the quantity of erythrocytes and leukocytes found in urine deposits Place one drop of urine deposit on a slide and cover with a coverslip. Table 7. b: degenerated cells. If many cells are present together with leukocytes and filaments.242 Manual of basic techniques for a health laboratory Fig. If a few are present.11 Ureteral and renal pelvic cells Note: Erythrocytes may be found in the urine of women if the specimen has been taken during the menstrual period.3 Reporting the results of microscopic examination of urine for erythrocytes Number of erythrocytes per microscope field 0–10 10–30 > 30 Result few erythrocytes (normal) moderate number of erythrocytes many erythrocytes . 7. c: pus. Report the results as described in Tables 7. shrunken. (c) pus: clumps of numerous degenerated cells.10 Leukocytes a: Intact cells. especially in clumps.11) Medium-sized oval cells with a distinct nucleus. Ureteral and renal pelvic cells (Fig. 10–15 mm (the nuclei may be visible).4.3 and 7. they may be from the ureter. examine the deposit and count the number of erythrocytes and leukocytes per microscope field. Fig. The presence of many leukocytes. less granular. Leukocytes (Fig. they may be cells from the renal pelvis. with no leukocytes.

13 Hyaline casts Renal cells (Fig. 7. 2) to the deposit.19). Renal cells are almost always present with protein in the urine.18). The nucleus is shiny and clearly visible. crossing almost the whole field when examined under the ¥ 40 objective. Fig. They are found in acute kidney disease. partly covered by amorphous phosphate crystals (b). 7. 7. The granules come from degenerated epithelial cells from the tubules of the kidney and have no diagnostic significance. Hyaline casts are transparent and slightly shiny.) Epithelial casts have no diagnostic significance. They may be found in healthy persons after strenuous muscular effort and have no diagnostic significance. 7.15) have smaller granules that do not fill the cast (a). Blood casts are filled with more or less degenerated erythrocytes. Epithelial casts are filled with pale yellow epithelial cells (Fig. 7. 7. the ends are rounded or tapered (Fig. 7. . Examination of urine 243 Table 7.17) are completely filled with leukocytes (a). the edges are indented and distinct and the ends are rounded (Fig. 7.7.14 Granular casts Pus casts (Fig.14).12) Renal cells are smaller than renal pelvic cells (the size of 1–2 leukocytes) and are very granular. 7. brownish in colour (Fig. Do not confuse with hyaline casts. They are soluble in ether but not in acetic acid.4 Reporting the results of microscopic examination of urine for leukocytes Number of leukocytes per microscope field 0–10 10–20 20–30 20–30 (degenerated) in clumps > 30 (degenerated) in clumps Result few leukocytes (normal) moderate number of leukocytes many leukocytes many leukocytes seen in clumps full field Fig.13).16). Casts Casts are cylindrical in shape and long. 7. Fatty casts are found in patients with severe kidney disease. Fine granular casts (Fig.12 Renal cells Fig. Fatty casts are very shiny yellowish casts. Pus casts are found in patients suffering from kidney infection. which may contain a few leukocytes (b). 7. Granular casts are rather short casts filled with large granules. Do not confuse with hyaline casts. (To make the cells more distinct. pale yellow in colour. add a drop of 10% acetic acid (reagent no. with rounded ends (Fig.

5% solution (reagent no. — aggregations of translucent mucus.15 Fine granular casts a: True fine granular casts. — grains of pollen from flowers (c). 0.20). Fig. Fig. b: hyaline casts. b: hyaline casts partly covered by amorphous phosphate crystals.17 Pus casts a: True pus casts.20 False casts a: Phosphate crystals. 7. 7.16 Blood casts Fig. 7.244 Manual of basic techniques for a health laboratory Fig. the following may be found (see Fig.18 Epithelial casts Fig. the ends tapering into threads (b). 7. False casts (Fig. 7.19 Fatty casts Fig. 7. 37)) (b). b: translucent mucus. Miscellaneous foreign substances If dirty receptacles or slides are used or if the urine specimen is left exposed to the air.21): — oil droplets (shiny) (a). 7. — starch granules (which will be stained blue–black with Lugol iodine. Do not mistake for casts: — clumps of phosphate crystals. . 7. — hairs (d). short and clear-cut (a).

c: pollen grains. very shiny.24) Size: 30–150 mm. 7. 7. which is made up of clumps of small granules with no definite shape (b). Crystals (Fig.23 Calcium oxalate crystals a: Envelope-shaped crystals. d: hairs. cubical or rose-shaped). Shape: envelope-shaped (a) or peanut-shaped (b). . b: peanutshaped crystals. 7. e: cotton fibres. b: starch granules. Shape: varies (square. Colour: colourless. Fig.25) Size: 30–150 mm.22) Crystals have regular geometric shapes (a). Fig. Fig. 7. Except in very rare diseases. 7. crystals in urine have no diagnostic significance. Shape: rectangular (a) or like a fern leaf or star (b). f: air bubbles. Triple phosphates (neutral or alkaline urine) (Fig. 7. Colour: yellow or brownish-red. Normal crystalline deposits Calcium oxalate (acid urine) (Fig. diamond-shaped.23) Size: 10–20 mm (a) or about 50 mm (b).7. 7. Examination of urine 245 Fig.21 Miscellaneous foreign substances a: Oil droplets. b: amorphous debris.24 Uric acid crystals — cotton fibres (e). Colour: colourless. shiny. unlike amorphous debris.22 Crystals a: Crystals. — air bubbles (f ). Uric acid (acid urine) (Fig. 7.

26) Size: about 20 mm.28 Calcium carbonate crystals Urates (alkaline urine) (Fig. Fig. b: needleshaped crystals. 7. Colour: colourless. Colour: yellow. whitish granules. 7. Fig.25 Triple phosphate crystals a: Rectangular-shaped crystals. Colour: colourless. Shape: similar to millet or corn grains. 7. the crystals dissolve. separate or in bundles. 7. Urates are often found together with phosphates. b: fern leaf-shaped crystals. 7. often scattered. shiny. Shape: like a star.26 Urate crystals a: Cactus-shaped crystals.27 Calcium phosphate crystals Fig. 7. 7.29) Size: 50–100 mm. grouped in pairs. giving off bubbles of gas.246 Manual of basic techniques for a health laboratory Fig. 7. Amorphous debris Amorphous phosphates (alkaline urine) (Fig. If acetic acid. Calcium carbonate (neutral or alkaline urine) (Fig. 7. . Shape: long prisms or flat blades. 2) is added. Shape: like a cactus (a) or a bundle of needles (b). Calcium sulfate crystals can be distinguished from calcium phosphate crystals by measuring the pH of the urine. 10% solution (reagent no.27) Size: 30–40 mm. Calcium sulfate (acid urine) (Fig.30) Amorphous phosphates appear as small. Calcium phosphate (neutral or alkaline urine) (Fig.28) Size: very small.

Examination of urine 247 Fig.31) Amorphous urates appear as very small. which are grouped in compact clusters.32 Cystine crystals They are soluble in acetic acid. Cholesterol (acid urine) (Fig. 7. When present. Shape: hexagonal plates. however. They are not soluble in acetic acid. 7. Cystine crystals are found only in fresh urine as they are soluble in ammonia. Cystine (acid urine) (Fig. but dissolve if the urine is gently heated. 2). 2) (one drop per drop of deposit). Colour: colourless.7.30 Amorphous phosphates Fig. 7.29 Calcium sulfate crystals Fig.32) Size: 30–60 mm. 7. They are found in patients with cystinuria. 10% solution (reagent no. Amorphous urates (acid urine) (Fig. . 10% solution (reagent no.33) Size: 50–100 mm. (Urine kept in the refrigerator often shows a heavy precipitate of urates. 7. with notches on one side. Cholesterol crystals are found in the urine of patients with nephrotic syndrome. they are found in large quantities in patients with certain diseases.31 Amorphous urates Fig. 7. a very rare hereditary disease. 7. Colour: colourless. very shiny.) Other crystalline deposits The following are rarely found in the urine. Shape: squarish plates. shiny. yellowish granules.

7. 7. Parasite eggs and larvae The following may be found: — eggs of Schistosoma haematobium: found together with erythrocytes (Fig.33 Cholesterol crystals Fig.35 Fungi Fig.248 Manual of basic techniques for a health laboratory Fig. Acetyl sulfonamide crystals are found in the urine following treatment with sulfonamide drugs.) Acetyl sulfonamides (neutral or acid urine) Shape: varied. 7.121): the urine appears white and cloudy. 7.34) Size: about 5 mm. Do not confuse with erythrocytes. Check that the urine specimen is fresh. Fungi (Fig. The presence of these crystals should be reported as they can cause kidney damage. 4. 7. Colour: brown.35) Size: 5–12 mm. — microfilariae of Wuchereria bancrofti (see Fig.36 Schistosoma haematobium Bilirubin (very rare) (Fig.36). but often similar to sheaves of needles. Budding may be seen. (The chemical test for bile pigments is positive. Shape: round or oval bodies of various sizes found together. Fungi are occasionally present in urine containing glucose. 7. Shape: square or like beads or needles. Fungi are not soluble in acetic acid. . 7.34 Bilirubin crystals Fig.

The first indirect evidence of Schistosoma haematobium infection is haematuria and/ or proteinuria. The filtration technique is used when quantitative information is required for epidemiological surveillance purposes. 2 ml of ordinary household bleach can be added to each 100 ml of urine.5% solution (reagent no.1) of at least 10 ml. If the urine cannot be examined for an hour or longer. 541 (or equivalent) filter-paper (filtration method) Conical flask for urine collection Pasteur pipettes (sedimentation method) Plastic syringe. as it may contain only a few ova.1). The sedimentation method is less sensitive but is cheaper and simpler to perform.2. Note: If formalin is not available. 12–20 mm pore size (nylon or polycarbonate) or Whatman No. The two methods used for detection of ova of Schistosoma haematobium are sedimentation and filtration.7. 15 ml (sedimentation method) Filter holder. Ask the patient to collect the urine in a clean flask or bottle. a 24-hour collection of terminal urine can be made (see section 7. q q q q q Method Collection of urine specimens The number of ova in the urine varies throughout the day. Microfilariae of Wuchereria bancrofti and Onchocerca volvulus may also be found in the centrifuged sediment of urine from patients in countries where filariasis is endemic. Examine the specimen at once. Alternatively. which is detectable using a urine dipstick (see section 7.8 Detection of Schistosoma haematobium infection In countries where schistosomiasis is endemic. The whole specimen must be examined. Gross haematuria indicates heavy infection.1. Trophozoites of Trichomonas vaginalis may also be seen. 37) (filtration method) Formaldehyde. Examination of urine 249 7. Materials and reagents q q q q q q q Microscope Microscope slides Coverslips Centrifuge (sedimentation method) Conical centrifuge tubes.2. The specimen should therefore be collected between these times and should consist of a single terminal urine specimen (see section 7. 10 ml (filtration method) Lugol iodine.2). Warning: Formalin and bleach are corrosive and must not be swallowed. This will preserve any eggs that might be present. 13 or 25 mm diameter (filtration method) Membrane filter. urine specimens are examined for eggs of Schistosoma haematobium.1. add 1 ml of undiluted formalin (37% formaldehyde solution) to each 100 ml of urine. . 0. 37% solution. it is highest in urine obtained between 10:00 and 14:00.

Fig.39 Drawing air into the syringe 5. Attach the syringe to the filter holder.38 Expelling the urine through the filter Fig. Important: — process the specimen as soon as possible. 7. Add one drop of Lugol iodine solution to improve the visibility of the eggs. carefully remove the membrane filter or filter-paper and place it on a microscope slide. Examine the entire filter under the microscope at ¥ 10 or ¥ 40. Filtration method 1. 7. The nylon membrane and filter-paper should be placed face-up. Allow the urine to sediment for 1 hour. 6. reattach the syringe to the filter holder and expel the air through the filter (Fig.37 Drawing urine into the syringe 4. Using forceps. 3. Fig. Place a filter in the filter holder. — label slides and tubes carefully. Disconnect the syringe from the filter holder. 2. Expel the urine from the syringe through the filter over a bucket or sink (Fig. Examine the deposit under the microscope for the presence of ova. Disconnect the syringe from the filter holder. 7.39).38). 3. Do not increase the centrifugation time and do not exceed 2000g as this may disrupt the ova and release miracidia.250 Manual of basic techniques for a health laboratory Sedimentation method 1. Centrifuge at 2000g for 2 minutes.37). 7. 7. Agitate the urine sample gently and draw 10 ml into the syringe (Fig. 7. 7. 7. . 2. Remove the supernatant and transfer the sediment into a centrifuge tube. Draw air into the syringe (Fig. — shake the container before pouring the urine specimen into the conical flask. while the polycarbonate membrane should be placed face-down. Shake the urine specimen well and pour into the conical flask. Record the results as the number of eggs per 10 ml of urine.40).

about 120–150 mm long. If no movement is seen. Heavy infection: > 50 eggs per 10 ml of urine. Check the filter under the microscope to ensure that it is free of parasites before reusing it. Examination of urine 251 Reuse of filters If you have used a plastic filter. Sometimes it is necessary to determine whether the eggs are viable. cystitis or nephritis).7). After soaking the filter. This is the most important part of the analysis. 7. in more than 10% of cases). or >1000 eggs per 10 ml of urine.7. such as >500 eggs per 10 ml of urine. wash it thoroughly with detergent solution. The urine is centrifuged at high speed and the resulting deposit is examined under the microscope (as described in section 7. one at each corner of the embryo. There are four flame cells. This can be done if the specimen is fresh and no preservatives have been added. remove it immediately after use and soak it overnight in a 1% hypochlorite solution (domestic bleach). Look carefully at the eggs to see if the embryos are moving. 7. Materials and reagents q q q q q q q q Fig.9 Detection of bacteria In healthy persons the urine contains practically no organisms. Microscopic examination The eggs of Schistosoma haematobium are large. look for the “flame cells” (Fig. Culture is always essential for precise determination of the identity of the organisms found and the quantity present. 7.g. b: flame cells. then rinse it several times with clean water. urethritis. An embryo (the miracidium) can be seen inside the egg.2. or where bacteria from an infection elsewhere in the body are excreted in the urine. the results may be reported according to egg count categories: q q Fig. However. the deposit may also be used to make smears that are stained by Gram and Ziehl–Neelsen stains and examined under the microscope. This is the best indication of viability. 7. may be appropriate in areas where the intensity of infection frequently reaches this level (i. Use a ¥ 100 objective with slightly reduced illumination to look for the rapid movement of cilia (short hairs) in the flame cells. and have a terminal spine at one end (Fig. Microscope Microscope slides Sterile 250-ml Erlenmeyer flask with stopper Centrifuge Sterile conical centrifuge tubes with stoppers Inoculating loop Bunsen burner or spirit lamp 70% Ethanol .41 (a)).40 Expelling air from the syringe Light infection: 1–49 eggs per 10 ml of urine.41 Schistosoma haematobium a: Miracidium.2.41 (b)). 7.e. A third category. Bacteria may be found in patients who have an infection of some part of the urinary tract (e. Reporting the results When the syringe filtration technique is used.

6.1) in the sterile flask. mix the deposit with distilled water until it forms a homogeneous suspension. 7.45(c)) Gram-positive fungi (Fig.42 Pouring off the supernatant fluid Fig. Seal the tube with either a screw-cap or a plug of sterile cotton wool fixed with gauze and string.3. Pour off the supernatant from the two tubes (Fig. 2. Microscopic examination Fig. Centrifuge the specimen at 1500g for 10 minutes. Pour 10 ml of fresh urine into a sterile centrifuge tube. 7. Collect a midstream specimen (see section 7. Examine the slide stained with Gram stain for the following (see section 5.) Preparation of slides 1.3). 7. If tuberculosis is suspected. 7. (Another way is to collect the urine in a conical tube rinsed only in boiling water and to examine it immediately.252 Manual of basic techniques for a health laboratory q Reagents needed for Gram staining (see section 5.3. Fig. 7.45(a)) Gram-positive cocci (Fig.44). prepare a smear from each of the two suspensions (Fig. Using an inoculating loop (sterilized by flaming) (Fig. using soap and water. centrifuge a further 10-ml specimen at 5000g for 20 minutes.1) and Ziehl–Neelsen staining (see section 5. 7.3. 7. Fix the slides by flooding with ethanol and flaming or by heating.44 Preparation of smears from the urinary deposit Examine the slides under the microscope using the ¥ 100 objective. 5.3. Examine as quickly as possible. 7. 7. .3).1): q q q q q pus (many leukocytes stained red by Gram stain) Gram-negative bacilli (Fig. 7.3.43 Mixing the urinary deposit 4. Using an inoculating loop (sterilized by flaming).45(d)).45(b)) Gram-positive diphtheroid bacilli (Fig. 3.1) and the second with Ziehl–Neelsen stain (see section 5.42).43).1. Stain the first slide with Gram stain (see section 5. Method Collection of specimens The genitals should be cleansed beforehand. Leave the slides to air-dry.

. Urine dipsticks Bacteria in urine may also be detected using urine dipsticks (see section 7. Tubercle bacilli appear dark red and are arranged in rows (Fig. A commercially available dipstick with reagents for the detection of nitrite (which is produced by certain pathogenic bacteria) and leukocyte esterase has been shown to have a high specificity and a high sensitivity for the detection of bacteria in urine. Reporting the results State whether pus or leukocytes are present. 7. 7. Gonococci Never diagnose a gonococcal infection on the basis of an examination of a urinary deposit.5).2). Examination of urine 253 Fig. c: Gram-positive diphtheroid bacilli. 7.46 Tubercle bacilli Examine the slide stained with Ziehl–Neelsen stain for tubercle bacilli.46). Fig.45 Bacteriological examination of urine a: Gram-negative bacilli. d: Gram-positive fungi. Look for gonococci in urethral pus (see section 5. b: Grampositive cocci.7.2. or Organisms found: — a few leukocytes — occasional erythrocytes — a few epithelial cells — a few Gram-negative bacilli. Example Organisms found: — many leukocytes — a few erythrocytes — a few epithelial cells — many Gram-positive cocci in clusters. Give a precise description of the organisms found.

a urine specimen should be dispatched to the bacteriology laboratory without delay for a semi-quantitative culture of the pathogenic organisms and for determination of their sensitivity to antimicrobials.254 Manual of basic techniques for a health laboratory Urine cultures Urine cultures are indicated when very high levels of bacteria are detected by microscopy or using urine dipsticks. In such cases. .

The stylet is withdrawn and the fluid flows freely through the needle (Fig. 8. The sterile lumbar puncture needle is inserted between the fourth and fifth lumbar vertebrae to a depth of 4–5 cm.1 Common reasons for investigation of CSF The most common reasons for investigating CSF are to exclude: — meningitis — bleeding into the central nervous system — certain cancers.3 Examination of CSF specimens 8. Tube 1 is used for visual inspection. microscopic and chemical analysis. Cells and trypanosomes are rapidly lysed in CSF samples. The volume of the CSF in adults is 100–150 ml. It is often caused by infection (see Table 8. It supplies nutrients to the tissues of the central nervous system and helps to protect the brain and spinal cord from injury. Examination of cerebrospinal fluid (CSF) 255 8.1 Location of CSF 8. 8.1). Glucose is also rapidly destroyed. The volume is less in children and varies according to the body length. Between 6 and 7 ml of CSF are collected in each of two tubes.1 Precautions q Do not delay in testing the CSF. Fig. the membranes lining the skull and covering the brain and spinal column.2 Collection of CSF specimens CSF specimens should be collected only by a physician or a specially trained nurse. numbered 1 and 2.3. Fig. 8. 8. Meningitis is an inflammation of the meninges. 2. Note: Immediate laboratory investigation of the CSF may be life-saving if meningitis is suspected.1).2). 26. 1. 8.8. unless preserved with fluoride oxalate (reagent no. see section 10.2 Collecting a CSF specimen 255 . 8. Leukaemia. Tube 2 is used for bacterial culture. tumours with manifestations in the brain and lead poisoning have also been shown to cause meningitis. Examination of cerebrospinal fluid (CSF) Cerebrospinal fluid (CSF) is contained in the cavity that surrounds the brain in the skull and the spinal cord in the spinal column (Fig.1).

Use pipettes plugged with non-absorbent cotton wool.256 Manual of basic techniques for a health laboratory Table 8.3 (b)). Often only a small quantity of CSF is available for examination. b: cloudy CSF. Never pipette CSF by mouth. 8. Fig.3 (c)). the CSF may appear slightly cloudy or greyish-white (Fig.. the CSF may appear cloudy and pink or reddish (Fig. especially S. q 8. The CSF may contain virulent organisms. or use a rubber safety bulb to draw up the fluid in the pipette.2 Direct examination Describe the appearance of the CSF specimen in the laboratory report.3 (a)). pneumoniae Staphylococcus spp.1 Common causes of meningitis Type of infection Bacterial Specific organism Neisseria meningitidis Streptococcus spp. Clear CSF Normal CSF is clear and colourless (Fig. 8. Cloudy CSF If pus is present. .3. Blood is usually present in the CSF for one of two reasons: — because of injury to blood vessels in the course of the lumbar puncture (in this case there is more blood in tube 1 than in tube 2). — because of a subarachnoid haemorrhage (in this case both tubes are the same colour). Protozoal Viral Plasmodium spp. 8.3 Examining the appearance of CSF a: Clear (normal) CSF. The specimen is difficult to collect so do not waste any of it. Mycobacterium tuberculosis Treponema pallidum Pseudomonas spp. Haemophilus influenzae Escherichia coli Listeria monocytogenes Leptospira spp. Bloodstained CSF If blood is present. Coxsackieviruses Arboviruses Echoviruses Polioviruses Mumps virus Arenaviruses Human herpesviruses Hepatitis viruses Fungal Candida albicans Cryptococcus neoformans q Work carefully and economically. c: bloodstained CSF. 8.

Xanthochromia Yellow discoloration of the CSF (xanthochromia.5 CSF from a patient with a subarachnoid haemorrhage Fig.5).3 Microscopic examination Microscopic examination of CSF includes: — examination of a wet preparation for blood cells.4). If the supernatant fluid is clear (Fig.6 CSF from a patient with xanthochromia . — examination of a Gram-stained smear for organisms that cause meningitis. such as Neisseria meningitidis. — examination of a Ziehl–Neelsen-stained smear if tuberculous meningitis is suspected. — to determine the total number of leukocytes (leukocyte number concentration). but clots may be found in the following diseases or conditions: — tuberculous meningitis: single or numerous small fine clots that can easily be overlooked. the blood is there because of a subarachnoid haemorrhage. If clots are present. wait for the erythrocytes to settle (or centrifuge at 2000g for 5 minutes) and examine the supernatant fluid. — examination of a wet preparation for trypanosomes in areas where African trypanosomiasis occurs. Fig. Fig. if suspected. Clot formation Examine the tubes of CSF 10 minutes after collection to see whether clots have formed. The above examinations are made using the deposit from centrifuged CSF. — to determine the types of leukocyte present (differential leukocyte count). the blood is there because of accidental injury to a blood vessel.1).6) may be caused by: — an old haemorrhage — severe jaundice — constriction of the spine. 8. — constriction of the spine: the CSF clots completely. Fig. If the supernatant fluid is bloodstained (Fig. Blood cells in the CSF The CSF may contain blood cells in varying quantities in certain diseases. 8. Normal CSF has no clots. Streptococcus pneumoniae and Haemophilus influenzae (see Table 8. — examination for fungi such as Cryptococcus neoformans and Candida albicans. The CSF is examined: — to detect erythrocytes. 8. 8.4 CSF from a patient with a blood vessel injury 8. 8. Examination of cerebrospinal fluid (CSF) 257 If only one tube of CSF is available. — purulent meningitis: a large clot. 8.3.8. they should be described in the laboratory report.

Make a 1 in 20 dilution using 0. If undiluted CSF is used. Gently mix the CSF and fill the chamber with the fluid (Fig.258 Manual of basic techniques for a health laboratory Important: The investigation of erythrocytes must be carried out as soon as possible after collection of the specimen. 2–5 ml Türk solution (reagent no. 8.2 mm. 8. Place the chamber on the microscope stage. since they are rapidly lysed. If a 1 in 20 dilution of CSF is used. Use of the Fuchs–Rosenthal counting chamber The Fuchs–Rosenthal ruled counting chamber has an area of 9 mm2 (modified chamber) or 16 mm2. 2.8 Covering the counting chamber with a coverslip Fig. Example: 150 cells per mm3 are reported as “150 ¥ 106/l”.9): — undiluted. using the ¥ 10 objective. 61). 8. the value does not change. examine the cells using the ¥ 40 objective to make sure that the cells are leukocytes. 8. The depth of the chamber is 0. Fig. Cover the counting chamber with the coverslip supplied (Fig.9 Filling the counting chamber with CSF Important: If undiluted CSF is used. Method 1. 3. 4. 8. When reporting in SI units. if the CSF appears cloudy. — diluted.8).7) q Microscope Fig. .10).05 ml of the CSF and 0. Determination of the leukocyte number concentration Materials and reagents (Fig. If erythrocytes are present. report as “number ¥ 106/l”. an improved Neubauer counting chamber may be used) Pasteur pipette with rubber teat Coverslips (supplied with the counting chamber) Bottle. the number of cells counted gives the number per mm3 of CSF. 8. Pipette into a small bottle and mix. no calculation is necessary. 13 and 16 (Fig.7 Materials for determining the leukocyte number concentration q q q q q Fuchs–Rosenthal counting chamber (if not available. 4. the number of cells counted is multiplied by 20 to give the number of cells per mm3 of CSF. 8. Leave the counting chamber on the bench for 5 minutes to allow the cells to settle. if the CSF appears clear. Count the cells in 1 mm3 of CSF. 7.95 ml of Türk solution. make the count using the ¥ 40 objective. Count the cells in 5 mm2 using squares 1.

10. Examine the cells under the microscope using the ¥ 40 objective. Make a thin smear and leave to dry.10. If there are many cells in the CSF: 1. 2. If undiluted CSF is used. 8. which is 9 mm3. Mix the deposit by tapping the end of the tube. pneumococcal): mostly neutrophils Tuberculous and viral meningitis: mostly lymphocytes Fig. Examination of cerebrospinal fluid (CSF) 259 Use of the improved Neubauer counting chamber If you are using an improved Neubauer chamber. mixed CSF on to a slide. Pour off the supernatant fluid into another tube (to be used for other tests).3.8. Examine the preparation under the microscope using the ¥ 40 objective. Centrifuge the CSF at 3000 g for 10 minutes. Fix with methanol and stain with a Romanowsky stain as described in section 9. Haemophilus influenzae. Pipette one drop of uncentrifuged. Spread on a clean slide and leave to dry. but Mott cells may be seen. as well as trypanosomes. 3. count the cells within the entire ruled area. If a 1 in 20 dilution of CSF is used. multiply the number of cells counted by 20 and divide by nine to give the number of cells per mm3 of CSF. 3.10. An increased number of leukocytes can be found in: q Bacterial meningitis (meningococcal.3. Determination of the leukocyte type number fraction (differential leukocyte count) q q q q q q q Materials and reagents Microscope Microscope slides Centrifuge Centrifuge tubes Pipettes Romanowsky stain (see section 9. Method If the CSF does not contain many cells (less than 200 ¥ 106/l): 1. Results Normal CSF contains less than 5 ¥ 106 leukocytes per litre (less than 5 per mm3). multiply the number of cells counted by 10 and divide by nine to give the number of cells per mm3 of CSF. . Fix with methanol and stain with a Romanowsky stain as described in section 9.1) Methanol. 2.10 Using the Fuchs–Rosenthal counting chamber q q African trypanosomiasis: mostly lymphocytes. Wet preparation for trypanosomes Method Place one drop of CSF deposit on a slide and cover with a coverslip.

in which the central nervous system is infected (see section 4. In a wet preparation stained with Romanowsky stain.11 Trypanosomes in a wet preparation stained with Romanowsky stain L: lymphocytes.13) Gram-positive Diplococci.7. 8.10. Culture of the organisms is necessary. numbers found. bacilli. etc. Neisseria meningitidis (meningococci) (Fig. 8. Report any organisms seen in the Gram-stained smear by their: q q q Gram reaction: positive or negative morphology: cocci.13 Streptococcus pneumoniae . Gram-stained smear for meningitis Method Make a smear of the CSF deposit and allow it to dry in the air. the leukocytes can be identified as lymphocytes (L).1. A definite species identification cannot be made from a Gram-stained smear only. lying side by side Intracellular. lying end to end q q Fig.5). These are large cells containing vacuoles and large amounts of immunoglobulin M (IgM) that stain dark with the eosin part of Romanowsky stains (see section 9. 8.11). inside the neutrophils.3. q q q Note: Diplococci may occasionally be seen outside the cells and are usually few in number. 8. T: trypanosomes. 8.3). The organisms that commonly cause meningitis are described on the following pages.12 Neisseria meningitidis Fig.3.260 Manual of basic techniques for a health laboratory The finding of motile trypanosomes in the CSF means that the later stage of trypanosomiasis has been reached. Fig. and Mott cells (M) can often be seen (Fig. diplococci. The protein concentration of the CSF is raised and the Pandy test is positive (see section 8.12) Gram-negative Diplococci.4). The fluid also contains an increased number of white blood cells. 8. Streptococcus pneumoniae (pneumococci) (Fig. M: Mott cells. Stain the smear with Gram stain as described in section 5.

i. 8. which is not visible with Gram stain Not intracellular Usually many in number. If organisms are seen (Fig. Examine the mixture between a slide and a coverslip. 8. fungi (Cryptococcus neoformans and Candida albicans) may be observed in a smear stained with Gram stain.3. 8.15 Acid-fast bacilli Fig. If a clot forms. 8. the CSF should be left to stand.3. Fig. it should be removed. 8. Method Mix on a microscope slide: — one drop of CSF deposit — one drop of Indian ink.17): — oval budding spores — short mycelium filaments. as described in section 5. Candida albicans may be found in an unstained wet preparation of CSF deposit.8.14) Gram-negative Small bacilli (coccobacilli) Not intracellular Often numerous. It appears as follows (Fig. Gram-positive bacilli Very rarely found.e. Culture is essential. Cryptococcus neoformans appears as follows (Fig. report the smear as “acid-fast bacilli present”. .16 Cryptococcus neoformans Fig. 2.3.16): — round budding spores containing greyish granulations. Fig. Ziehl–Neelsen-stained smear for tuberculous meningitis Method If tuberculous meningitis is suspected. In patients with meningitis (especially purulent and tuberculous meningitis). spread on a slide and stained with Ziehl–Neelsen stain. 8.14 Haemophilus influenzae In all the above-mentioned forms of meningitis the leukocytes present are neutrophils. May belong to the Listeria group.4 Determination of glucose concentration Glucose concentrations in the CSF are normally about 60% of those in blood. the concentration of glucose in the CSF is greatly reduced.17 Candida albicans 8.2 mmol/l (45–75 mg/100 ml). 8.5–4. Examination of cerebrospinal fluid (CSF) 261 q q q Surrounded by a capsule. Fungi in the CSF Very rarely. Cryptococcus neoformans may be found in cloudy CSF with lymphocytes. q q q q Haemophilus influenzae (especially in young children) (Fig. — each group of 1–3 spores is surrounded by a colourless capsule.15). 8.

3% solution (reagent no. Method for determination of globulin (Pandy test) 1. 2. 8. 57) Pandy reagent (reagent no. Compare the cloudiness of the test sample against the protein standards (Fig. .5). The normal concentration of protein in the CSF is 100–450 mg/l. 3. 8. four times more CSF is needed than in the test on blood. 41) Protein standards (see section 7. Important: As the glucose in the CSF is rapidly destroyed once the fluid is collected. Pipette 3 ml of sulfosalicylic acid into a test-tube that matches the standard tubes. all methods that are used for determination of blood glucose concentrations can be applied. it is important to carry out the estimation of glucose concentration as soon as possible.5 Determination of protein concentration Principle The total protein concentration in the CSF is measured by diluting the CSF in sulfosalicylic acid and comparing the cloudiness produced against a set of protein standards. Place the tube in front of a piece of black cardboard.18) q CSF: centrifuge the CSF at 2000g for 5 minutes and use the supernatant fluid Graduated pipettes Dropping pipettes Test-tubes Test-tube rack Black cardboard Sulfosalicylic acid. 8. q q q q Fig. 8.19 Comparing a test sample against the protein standards 2.1) is used. Leave the tube for 5 minutes. A raised globulin level in the CSF is shown by adding the CSF to a phenol solution in the Pandy test (see below).262 Manual of basic techniques for a health laboratory Method For determination of glucose concentrations in the CSF. Add 1 ml of clear CSF supernatant fluid and mix.3. 8. Fig. The protein concentration is increased in: — meningitis. 26). If there is likely to be a delay. When the orthotoluidine method (see section 10. — African trypanosomiasis. Measure 1 ml of Pandy reagent into a small test-tube.2.19). subarachnoid haemorrhage or constriction of the spine.18 Materials and reagents for determining the protein concentration of CSF q q q q Method for determination of total protein 1. Materials and reagents (Fig. Record the concentration of protein in the CSF in g/l. the CSF should be preserved in fluoride oxalate (reagent no.

mainly lymphocytes Elevated. 8. yellowish Normal or slightly elevated Highly elevated 3. 1–10 g/l Elevated Normal or slightly elevated Elevated Elevated Not interpretable Glucose concentration Greatly reduced Greatly reduced Normal Reduced Reduced Not interpretable Normal Other findings Bacteria Bacteria. mainly granulocytes > 5 cells/ml. mainly granulocytes 30–300 cells/ml.3. If globulin is present. Do not put it in the refrigerator. 56).4 Dispatch of CSF specimens for culture Before dispatch keep the CSF in the incubator at 37 °C.20 Adding CSF to Pandy reagent Fig. If globulin is absent.1 Materials and reagents q Flat bottles containing an appropriate transport medium. Read the results immediately.20). . a white cloud forms as the drops of CSF mix with the reagent (Fig. 8. Examine the solution after the addition of each drop. or there is a slight cloudiness that redissolves (Fig. Report the test as “Pandy test positive” or “Pandy test negative”. modified (reagent no.8. 8. 4. Mott cells After centrifugation. b: negative result (b) Table 8.6 Summary Table 8. 8.21 Pandy test for globulin a: Positive result. slowly add three drops of CSF (Fig.21 (a)). clotted proteins — — Trypanosomes. Using a dropping pipette. 8. mainly lymphocytes 10–300 cells/ml. such as Stuart transport medium.2 summarizes the typical findings on examination of CSF.2 Typical findings on examination of CSF Disease or condition Purulent meningitis Tuberculous meningitis Viral meningitis Malaria African trypanosomiasis Subarachnoid haemorrhage Compression of the spine Appearance Cloudy. Examination of cerebrospinal fluid (CSF) 263 (a) Fig. 8. mainly lymphocytes Not interpretable Protein concentration Highly elevated. red — Clear. yellowish Clear or almost clear Clear Slightly cloudy Clear or slightly cloudy Red Blood cell concentration > 3000 cells/ml. 8.21 (b)). no white cloud forms as the drops of CSF mix with the reagent.4. 8.

8. The medium is supplied in 30-ml bottles that contain 8 ml of solid medium (along one side of the flat bottle). Fig. If possible. 8.22).264 Manual of basic techniques for a health laboratory 8.4.22 Dispatching CSF specimens for culture using Stuart transport medium Preservation time: up to 4 days at room temperature. otherwise use uncentrifuged CSF. sow centrifuged CSF deposit on the medium (Fig. The bottles are filled with a mixture of air (90%) and carbon dioxide (10%). .2 Method using Stuart transport medium (for the isolation of Neisseria meningitidis) This is the best method.4. Follow the instructions given for gonococci in section 5.5.

2 Leukocytes 265 .9. and no risk in taking two 10-ml tubes or more for analysis.1) Appearance: round or slightly oval cells filled with haemoglobin. 9. They can be identified by microscopy after staining with a Romanowsky stain (see section 9. they appear pink with a pale central area.1 Types of blood cell Three main classes of blood cell can be distinguished under the microscope: red cells (erythrocytes).5 litre of blood as a donation for transfusion. thus removing the principal end-product to which most organic substances are metabolized in the body. Number concentration: normally around 4–5 ¥ 1012 per litre (4–5 ¥ 106 per mm3) of blood. 9. There is therefore no danger involved in taking 0. Fig. 9. lymphocytes and monocytes) which differ in size. 9. 9.1.2) Appearance: round cells containing a nucleus and granules in the cytoplasm. They also carry carbon dioxide from the tissues to the lungs. Number concentration: normally about 8 ¥ 109 per litre (8000 per mm3) of blood. There are five types of leukocyte (neutrophils. colour of the granules in the cytoplasm and other factors.4). Volume of blood in the human body An adult weighing 60 kg has about 4.1 Erythrocytes (Fig. Make this clear to anxious patients when you take their blood. Size: 9–20 mm. 9. eosinophils. basophils.1.10.4). Fig.5 litres of blood.1 Erythrocytes 9.10. The presence of a nucleus enables leukocytes to be readily distinguished from erythrocytes under the microscope. Size: 7–8 mm. Leukocytes play an important role in the defence or immune system.2 Leukocytes (Fig. Haematology Haematology is the study of the cells that are found in blood and the factors that affect their functioning. After staining with a Romanowsky stain (see section 9. white cells (leukocytes) and platelets (thrombocytes). Erythrocytes carry haemoglobin which combines with and carries oxygen from the lungs to the tissues. they do not contain nuclei. shape of the nucleus. Haematology 265 9. From the side erythrocytes look like biconcave discs.

Cytoplasm: very little visible.4): — the serum. a yellow liquid. Blood treated with an anticoagulant separates into two liquid components (Fig. 9.2% solution (reagent no.5): — the plasma. clotting is prevented and the blood remains fluid. 3. Fig. Size: 2–5 mm. trisodium citrate. Clotted blood separates into two components (Fig. 60) and EDTA dipotassium salt.1. Serum does not contain fibrinogen. 22).266 Manual of basic techniques for a health laboratory 9.3 Thrombocytes Clotting of blood1 When blood is collected in a glass tube it solidifies within 5–10 minutes forming a clot. but all the other proteins are present.5 Blood treated with an anticoagulant 1 See section 9. — the clot. contains granules. which sediment over time or following centrifugation to form a thin layer of leukocytes over a deposit of erythrocytes.9. In healthy adults. Difference between plasma and serum q Plasma contains a soluble protein called fibrinogen in addition to a large number of other proteins. If an anticoagulant is added to the blood as soon as it is collected. 9. 9. 10% solution (reagent no.4 Clotted blood Fig. a solid red mass. Examples of anticoagulants include: fluoride oxalate (reagent no. 26). . 9. The fibrinogen is changed into insoluble fibrin. q Fig. 9. 9.3) Thrombocytes or platelets are fragments of megakaryocytes that are found in the peripheral blood. the blood contains about 150–300 ¥ 109 thrombocytes per litre (150 000–300 000 per mm3).3 Thrombocytes (Fig. which together with the erythrocytes forms the clot. where they are involved in clot formation. it has coagulated. a yellow liquid. — the blood cells.

as described below. the ear or the heel (in infants). as described in section 9. (For example. Keep a stock of sterile needles in a small glass tube: the point should rest on a pad of non-absorbent cotton wool and the tube should be plugged with the same material. 10 ml. q q Fig.3 Method Preparation 1.7).9. Lay the patient’s arm on the table.2 Collection of blood specimens 9. either empty or containing an anticoagulant (see section 9. (b) Prepare the correct bottle or tube to be used for each test.8). the first 1 ml of blood must be discarded when blood is taken for coagulation assays.1. palm upwards. 2.6 Materials for venepuncture Fig. Before taking the blood. 30–40 mm. 9. Read the patient’s request form carefully: (a) Decide how much blood is needed. Ask the patient to sit alongside the table used for taking blood. 5 ml. Capillary blood may be collected from the finger. 2 ml. 9.4. 20 gauge. at the 5-ml level). 9.1 Principle Venous blood is collected from a vein in the arm with a needle and syringe. medium bevel For collection of blood: — syringes.2. plan the sequence in which blood samples must be taken. 19 gauge. 2–3 mm bore — needles. 18 gauge. 9. Haematology 267 9.) 3.g.2. and support it by placing a small cushion under the elbow (Fig. . 9.2 Materials and reagents q For disinfecting the skin: — cotton wool — 70% ethanol or tincture of iodine For the venepuncture (Fig. 20 ml (check that the end of each syringe fits into the needle) — bottles or test-tubes (Fig. If blood is to be used for different laboratory investigations. 9.7 Bottles and test-tubes for collection of blood specimens If blood samples are to be taken from children under 5 years. 9.6): — gloves — a tourniquet of soft rubber tubing.1.3) and bearing a mark corresponding to the required amount of blood (e. wash your hands with soap and water. 23 gauge or 25 gauge needles will also be required.2.

9. 9. 9. The correct site to take the blood is the vein in the bend of the elbow. If possible. Test the needle and syringe to make sure that the needle is not blocked and the syringe is airtight. Apply the tourniquet.10 Sites for taking venous blood 1: Preferred site. lay his or her arm in an outstretched position (Fig. 9.11 Applying a tourniquet Fig. If necessary. 9. Procedure 1. 4: alternative sites. 3.9 Taking blood from a patient in bed If the patient is in bed. The tourniquet should be just tight enough to slow down the blood flow in and distend the veins. Fig.12 Tying a tourniquet . 4. Loop the end under the main part of the tourniquet (Fig. 9. but it must not be so tight that the blood flow in the arteries is reduced. Place the end of the needle in the sterile tube until ready for use.9). Fig. at the point where the vein is thickest and most easily visible (Fig. Fix the needle on to the syringe.8 Taking blood from a patient in the laboratory Fig. points 2.268 Manual of basic techniques for a health laboratory Fig. 3 and 4 can be used as alternatives. 3. 5.12). 2.10).11). pull one of the ends across (Fig. wrap the tourniquet firmly round the arm and hold the ends. Ask the patient to open and close his or her hand several times to swell the veins. 9. With the left hand. 9. 9. 2. With the right hand. touching only the top of the needle. choose one of the branches forming a Y just above their junction (1).

0– 1. 10. 9. 8. Withdraw the needle in one rapid movement from under the swab (Fig. Take the syringe in your right hand. Then continue to withdraw the piston to fill the syringe with the required amount of blood (Fig. Blood should appear in the syringe (Fig.14 Correct position for holding a syringe Fig. 7.15) without hesitation. feel for the vein where you will introduce the needle (Fig.17 Checking the needle is inserted correctly . 9. 9. Push the needle along the line of the vein to a depth of 1.9. which is resistant. Haematology 269 Fig. 9.16). 9.19). Position the needle with the bevel uppermost. 9. 9.15 Correct position for venepuncture Fig.13 Feeling for a vein Fig. Apply a dry swab over the hidden point of the needle. Make the venepuncture entering the centre of the vein (Fig.14). Disinfect the skin with a swab dipped in tincture of iodine or ethanol. 9. 12. holding your index finger against the top of the needle (Fig. 9. — then the wall of the vein. 11.17). Using the index finger of your left hand. 9. Remove the tourniquet by pulling on the looped end. which is less resistant (more flexible).18).5 cm. Fig.16 Incorrect position for venepuncture 6. With your left hand pull back the piston of the syringe slowly. 9. 9. Important: Never approach a vein from the side (Fig. You will feel the needle going through: — the layer of skin.13). 9. 13.

21).19 Withdrawing the needle 14.20 Correct (a) and incorrect (b) positions for stopping blood flow . Ask the patient to press firmly on the cotton wool swab for 3 minutes. 9. keeping the arm outstretched (Fig. 16. Fig.4).5. Remove the needle from the syringe. Label the tubes or bottles clearly with: — the patient’s name — the date — the patient’s outpatient or hospital number if this is available. 9.20(a)). Place the rinsed needles and syringes in small glass tubes plugged with nonabsorbent cotton wool and sterilize in the autoclave or the dry-heat sterilizer (see section 3. then rinse in disinfectant (see section 3. Fill the specimen tubes or bottles with the blood up to the mark (Fig.18 Filling the syringe with blood Fig. Bending the arm back over the swab (Fig. as they cannot be resterilized. Disposable needles must only be used once.5).20(b)) is not recommended (because of the risk of a haematoma). 15. Immediately invert the tubes or bottles that contain anticoagulant several times. 9. 9. Rinse the needle and syringe at once with cold water. 9.5.270 Manual of basic techniques for a health laboratory Fig. 9. Never use a needle or syringe on another person before it has been resterilized.

some laboratories are using the unit “gram per litre” (g/l).062 = haemoglobin(Fe) 9. it is necessary to specify the chemical structure to which it applies. Example: haemoglobin 150 g/l ¥ 0. Haematology 271 9.062. as an interim measure. It should be used wherever possible. The haemiglobincyanide photometric method gives the most accurate haemoglobin estimations. Units of measurement The SI unit for expressing haemoglobin concentrations is millimole per litre (mmol/ l).1 Haemiglobincyanide photometric method Principle The blood is diluted in Drabkin diluting fluid. and it is not necessary to say “haemoglobin(Fe)”.3.3 mmol/l In this manual calculations and values are usually expressed in both forms. UNICEF Plads. DK 2100 Copenhagen. Values in grams per litre may be converted into values in millimoles per litre by multiplying by 0.0 g/100 ml. the simple term “haemoglobin” suffices. From such a curve a graph can be prepared and a table made for the haemoglobin values.98 (110 V battery) or 09. which may be: — the fresh haemiglobincyanide reference solution used to calibrate the instrument. The solution obtained is examined in a spectrophotometer (or colorimeter).2 ml Drabkin diluting fluid (reagent no. . 09. However.310.309. before making the change to millimole per litre.21 Transferring the blood to a specimen tube 9. When this unit is used. — a reference solution previously calibrated against the haemiglobincyanide reference solution. 1 Some spectrophotometers run either on mains electricity or on current from a motor car battery. this means that the term “haemoglobin(Fe)” should be used instead of the simple term “haemoglobin”.3 Estimation of the haemoglobin concentration Haemoglobin is the red pigment contained in erythrocytes. which haemolyses the red cells and converts the haemoglobin into haemiglobincyanide (cyanmethaemoglobin). it can be ordered from the following address: UNICEF. or — a blood sample of known haemoglobin concentration. When this unit is used. 21) Reference solution. 9. Its absorbance is proportionate to the amount of haemoglobin in the blood. Materials and reagents q q q q q q q Spectrophotometer1 (or colorimeter) Spectrophotometer (or colorimeter) cuvettes Test-tubes Test-tube rack Blood (Sahli) pipettes. Denmark. Fig. 0. One model is supplied by UNICEF: reference no. the values are 10 times greater than values in the traditional unit “gram per 100 ml”. In practice. It should be noted that if the unit “gram per litre” is used. For example.00 (220 V battery). It consists of protein chains and iron-containing molecules.9. A calibration curve must be prepared before the spectrophotometer (or colorimeter) can be used for haemoglobin estimation. 150 g/l = 15. Freeport.

3 1.02 ml of blood and 4 ml of Drabkin diluting fluid). 9.22). Fig. Pipette into each tube the amounts shown in Table 9.0 Dilution undiluted 1:2 1:3 1:4 1 If a dilution of 1 in 200 is used (i. Since 10 ¥ 251/1000 is very nearly 2.5. 9. 3.e. c = the factor for converting milligrams to grams.0 2.5 = 150 g/l 2. Calculate the haemoglobin value of the reference solution in grams per litre by using the following formula: concentration in mg 100 ml ¥ 10 a ¥ 251b 1000 c where: a = the factor for converting 100 ml to 1 litre.02 ml of blood is diluted with 5 ml of Drabkin diluting fluid.0 instead of 2. 0. Read a reference solution (see above). (b) Fill a matched cuvette with Drabkin diluting fluid and place in the spectrophotometer (or colorimeter). 9. .272 Manual of basic techniques for a health laboratory Important: At the beginning of each day: q q Clean the matched spectrophotometer (or colorimeter) cuvettes. Read the dilutions in the spectrophotometer (or colorimeter): (a) Set the wavelength to 540 nanometres (nm) or place a green filter in the spectrophotometer (or colorimeter). Prepare a series of dilutions of the reference solution in four test-tubes (labelled 1–4) (Fig. Mix the contents of the tubes and allow to stand for 5 minutes (Fig. the above formula can be simplified as follows:1 haemoglobin value of reference solution in grams per litre = concentration in mg/100 ml ¥ 2.7 3. b = the dilution factor when 0.0 2.0 2. which is used to zero the spectrophotometer (or colorimeter).0 1. multiply by 2. 4.23). q Calibration of the spectrophotometer (or colorimeter) using haemiglobincyanide reference solution (or a reference solution previously calibrated against haemiglobincyanide reference solution) 1.1 Preparing serial dilutions of reference solution Tube number 1 2 3 4 Volume of reference solution (ml) 4. Fill one of the cleaned tubes with fresh Drabkin diluting fluid.0 Volume of Drabkin diluting fluid (ml) 0.5.22 Preparing serial dilutions of haemiglobincyanide reference solution Table 9.1.5 Example: concentration of reference solution = 60 mg/100 ml haemoglobin value = 60 ¥ 2.

Prepare a graph. Switch on the spectrophotometer (or colorimeter) and set to wavelength 540 nm.5 Fig. Pipette 8 ml of Drabkin diluting fluid into a test-tube. 9. 7.3. Zero the spectrophotometer using Drabkin diluting fluid. leave them to stand for 5 minutes Fig. 6. 2. 5.5 8.23 After mixing the dilutions of reference solution. (d) Read the contents of tubes 1 to 4. From the graph make a table of haemoglobin values from 20 to 180 g/l. Be sure to wipe the outside of the pipette beforehand to avoid adding excess blood. 4.2 and Fig.2 Sample spectrophotometer readings for different dilutions of reference solution Dilution undiluted 1:2 1:3 1:4 Haemoglobin concentration (g/l) 150 150/2 = 75 150/3 = 50 150/4 = 37. 5.5 11. Haematology 273 Table 9. Add 0.9. Prepare a series of dilutions of the haemiglobincyanide solution in four testtubes (labelled 1–4) as shown in Table 9.g. Read and record the absorbance of the haemiglobincyanide solution prepared above. Leave to stand for 10 minutes. 9.5 Absorbance at 540 nm 35. Read and record the absorbances of the diluted solutions. plotting the readings of the diluted reference solutions against their respective haemoglobin concentrations (Table 9. Mix the haemiglobincyanide solution by inverting several times. 168 g/l).24). Obtain a sample of blood of known haemoglobin concentration (e. using a cuvette.04 ml of well-mixed blood. Calibration of the spectrophotometer (or colorimeter) using a blood sample of known haemoglobin concentration 1. 6.24 Standard curve for determining the haemoglobin concentration of blood specimens (c) Zero the spectrophotometer.0 17. 9. Make sure the needle returns to zero between each reading with Drabkin diluting fluid. 3. .

the haemoglobin estimation must be carried out within 6 hours. Store Drabkin diluting fluid in a brown reagent bottle because it decomposes on exposure to light.0 3. use a clear glass bottle carefully wrapped in silver foil.4 In this example. Plot a graph of absorbance against haemoglobin concentration. Check that the blood is still on the 0. Pipette 5 ml of Drabkin diluting fluid into a tube.0 2. Wash your hands immediately after handling it. It must be kept in a locked cupboard at all times when not in use. Do not freeze. it should be discarded. as this may cause decomposition of the compound.7 3. q q q q Method 1.25). Wipe the outside of the pipette. 0. Draw venous or capillary blood to the 0. Determine the haemoglobin concentrations for each of the absorbances from the graph.0 Volume of Drabkin diluting fluid (ml) 1.02 and ending at 1.4 10. 9. or loses its colour. 3. A reference table of values is prepared using the graphs obtained from either of the above methods: q Draw up a table of absorbance readings starting from 0. If a brown reagent bottle is not available. The absorbance must read zero.01. Mix the contents of the tube and leave for 5 minutes (see Fig. Drabkin diluting fluid remains stable for several months when stored at cool temperatures. Squeeze the bulb of the pipette to expel the blood into the Drabkin diluting fluid and rinse the pipette by drawing up and expelling the fluid in the tube three times. Draw a straight line starting at the origin passing as close to each point as possible.50.0 4. . If the room temperature exceeds 30 °C.274 Manual of basic techniques for a health laboratory Table 9. 2.1 6.0 1. If it becomes turbid. The clarity of the diluting fluid can be checked by measuring its absorbance in a spectrophotometer at 540 nm against water as a blank. it is assumed that the haemoglobin concentration of the haemiglobincyanide solution is 168 g/l.3 Preparing serial dilutions of haemiglobincyanide solution Tube number 1 2 3 4 a Volume of haemiglobincyanide solution (ml) 4.0 3.23). Once the haemiglobincyanide solution has been prepared.02-ml mark of a blood (Sahli) pipette. 9. q Precautions q Potassium cyanide is very poisonous. using ordinary graph paper. Always allow the diluting fluid to warm to room temperature before use.02-ml mark (Fig. store it in a refrigerator at 4–6 °C.0 Concentration of haemoglobina (g/l) 13. With venous blood ensure that it is well mixed by inverting the bottle containing it and the anticoagulant repeatedly for about 1 minute immediately before pipetting it. Do not allow air bubbles to enter. 0. Extend the line so that you can read absorbances for haemoglobin values greater than 168 g/l. Drabkin diluting fluid should be clear and pale yellow.00. 8.0 2.

2 Haemoglobin concentration (g/l) 136–196 113–130 115–148 120–160 130–180 .4–9.9 8. Haematology 275 Fig.1–11. by age group Age group Newborn infants Infants (1 year) Children (10–12 years) Women Men Haemoglobin (Fe) concentration (mmol/l) 8. Centrifuge the diluted blood at 2000g for 5 minutes before taking a reading.4–12.1 7.2 7.25 Checking that the blood is still on the mark 4. record the concentration of haemoglobin in g/l. 9.9. this may be attributable to abnormal plasma proteins or to a high concentration of white cells.4 shows the reference ranges for different age groups.1 7. Zero the colorimeter using Drabkin diluting fluid. Reference range Table 9.4–9. Using the table prepared from the calibration curve.4 Normal haemoglobin concentrations. Read the absorbance of the patient’s diluted blood in the spectrophotometer test-tube or cuvette. If cloudiness appears in the diluted blood.0–8. Table 9.

haemoglobinometer or colorimeter Test-tubes Test-tube racks Corks or rubber stoppers Cuvettes Grease pencil Cotton wool or gauze AHD standard (supplied by the central laboratory) AHD reagent (reagent no. Replace the undiluted AHD reagent in the cuvette with the diluted AHD standard solution. 3. 5. e. Fill a clean cuvette with the undiluted AHD reagent. The AHD reagent can be prepared using chemicals that are generally available locally. which is a stable coloured compound. a stable crystalline compound that is commercially available. 160 g/l at a 1:150 dilution. which is highly toxic.3. The absorbance of the alkaline haematin D-575 is measured using a haemoglobinometer or colorimeter. The calibration procedure uses chlorhaemin.g. the haemoglobin is converted to alkaline haematin D-575. 8).g. q q Materials and reagents q q q q q q q q q Spectrophotometer. in contrast to Drabkin diluting fluid for the haemoglobin cyanide method. 2. The AHD reagent does not include potassium cyanide. Dry the outside of the cuvette with cotton wool or gauze and place it in the cuvette chamber. Note the concentration of the AHD standard indicated on the label. 4. whereas with a colorimeter. Stopper the test-tube using a clean cork or rubber stopper and mix by inversion. but less expensive. Leave the tube to stand for 2–3 minutes. Adjust the spectrophotometer or haemoglobinometer to read zero (blank). . repeat the measurement procedure and adjust the spectrophotometer or haemoglobinometer to read the correct haemoglobin concentration indicated on the label. The spectrophotometer and haemoglobinometer directly determine the haemoglobin (Hb) concentration of the blood sample. 160 g/l.2 Alkaline haematin D method Principle When a blood sample is added to an alkaline solution containing a non-ionic detergent. Pipette 20 ml of AHD standard into a clean test-tube containing 3 ml of AHD reagent. the haemoglobin concentration of the blood sample is obtained from the absorbance using a prepared calibration curve or table of values.276 Manual of basic techniques for a health laboratory 9. Calibration of the spectrophotometer or haemoglobinometer 1. e. The alkaline haematin D (AHD) method offers several advantages over the haemoglobin cyanide method: q q It is as accurate.

Pipette 5 ml of AHD reagent into the test-tube marked B. draw a straight line joining through as many of the points as possible. type of cuvette. Stopper each tube and mix by inversion.9. Starting from the origin. Switch on the spectrophotometer or haemoglobinometer. close the cuvette chamber and adjust the colorimeter to read zero absorbance (blank). Replace the AHD reagent in the cuvette with the reference solution from test tube 4. Pipette the indicated volumes of AHD reagent and reference solution into testtubes 1–4. B and N. Method Method using a spectrophotometer or haemoglobinometer 1. Arrange six test-tubes in a test-tube rack. 6. 9. 3. Switch on the colorimeter and set the wavelength to 540 nm. 2. Dilute the reference solution in test-tube N as described in Table 9. 4. Dry the outside of the cuvette with cotton wool or gauze. 9. 11.5 Preparation of serial dilutions of AHD reference solution for calibration of a colorimeter Test-tube AHD reagent (ml) AHD reference solution (ml) Total volume (ml) 1 1 4 5 2 2 3 5 3 3 2 5 4 4 1 5 . 2. or method for haemoglobin measurement. Note: Always prepare a new calibration curve whenever you use a different colorimeter. Pour the solution back into test-tube 4. 5.26). Calculate the haemoglobin concentrations in the test-tubes as follows: haemoglobin concentration = concentration of reference solution ¥ dilution factor For example: tube N: 160 g Hb/l tube 1: 160 g Hb/l ¥ 4/5 = 128 g Hb/l tube 2: 160 g Hb/l ¥ 3/5 = 96 g Hb/l tube 3: 160 g Hb/l ¥ 2/5 = 64 g Hb/l tube 4: 160 g Hb/l ¥ 1/5 = 32 g Hb/l tube B: 0 g Hb/l 8. 10. respectively in sequence. Allow the colorimeter to warm up for the time recommended by the manufacturer. 7. Plot a graph of the absorbance values against the haemoglobin concentration (g/l) for the standard and test samples (N and tubes 1–4. Label the test-tubes 1. Pipette 3 ml of AHD reagent and 20 ml of AHD standard into the test-tube marked N. Place the cuvette into the cuvette chamber. Pour the AHD reagent from test-tube B into a clean cuvette.5. 1 and N. 4. Table 9. Repeat the procedure using test-tubes 3. 2. Record the absorbance. respectively) (Fig. Allow it to warm up for the time recommended by the manufacturer (usually 10 minutes). 3. Haematology 277 Calibration of the colorimeter 1.

Flush the pipette carefully five times with the AHD reagent. Repeat the procedure with the solution in test-tubes C1 and C2. Make sure that there are no air bubbles in the solution. Arrange the test-tubes in a test-tube rack: one for each sample to be tested. Pipette 20 ml of AHD standard into test-tubes C1 and C2. Results Report the results in g/l. However. and C1 and C2 for the control samples.5%. Pipette 3 ml of AHD reagent into each test-tube. Method using a colorimeter The AHD method is also applicable using a colorimeter. Dry the outside of the cuvette with cotton wool or gauze. Pour the AHD solution from test-tube B into a clean cuvette. 6. 7. B for the blank. 4. Pipette 20 ml of blood collected in EDTA from a patient into the AHD reagent of the appropriate tube. Place the cuvette in the cuvette chamber and adjust the spectrophotometer or haemoglobinometer to read zero. Therefore. Record all the results. If the readings of the two controls differ by less than 2. one for the blank and two for the control samples. measure the haemoglobin concentration of all the test samples. as described above. Leave the tubes to stand for 2–3 minutes. 9. label the test-tubes with the appropriate laboratory numbers of the samples to be measured. . Using a grease pencil. respectively. 3. a calibration curve must be used to relate the absorbance readings to the haemoglobin concentration. Plug all the test-tubes with a clean cork or rubber stopper and mix by inversion. the absorbance in a colorimeter does not increase linearly with haemoglobin at elevated concentrations.26 Calibration carve for determining haemoglobin concentration 2.278 Manual of basic techniques for a health laboratory Fig. Example: “haemoglobin = 89 g/l”. The measurement procedure is the same as that described for a spectrophotometer or haemoglobinometer. 9. 5. 8.

Faulty technique: — using a dilution factor different from the one for which the spectrophotometer. and the result is a simple decimal fraction with no unit).4 Estimation of the erythrocyte volume fraction The total volume of erythrocytes in a given volume of blood divided by the volume of blood is called the erythrocyte volume fraction. The erythrocyte volume fraction is therefore a measure of the proportion of erythrocytes to plasma. — insufficient mixing of venous blood. — dirty pipettes. — excessive squeezing of the finger after pricking. send the machine to a servicing agent. If the latter are ignored. haemoglobinometer or colorimeter requires frequent recalibration. For example. the unit “ml” cancels out. every 2–3 days. erythrocyte volume fraction plus plasma volume fraction = 1). Faulty or dirty equipment. — air bubbles trapped in pipettes. which has sedimented after collection. — dirty cuvettes.9. — adding too little or excess blood to Drabkin diluting fluid.0.g. — a defective spectrophotometer. — air bubbles in the cuvette.55 (note that 0. haemoglobinometer or colorimeter. Haematology 279 Errors in haemoglobin estimation Errors in sampling: — inadequate flow of blood from the finger prick.45 + 0.55 = 1. the erythrocyte volume fraction is 450 ml/1000 ml = 0. — dirty filters. — placing the cuvette in the chamber with the frosted sides facing the light path.e. — using the wrong filter for the colorimeter. The remainder of the blood is made up almost entirely of plasma. — prolonged use of a tourniquet when collecting venous blood. 9. such as: — broken or chipped pipettes. change the bulb and repeat the procedure for internal quality control. — small clots in venous blood due to inadequate mixing with EDTA after collection. i. If the problem of frequent recalibration persists. if the volume of erythrocytes in 1 litre (1000 ml) of blood is 450 ml. . — inadequate mixing of reagent.45 (since the fraction is millilitres divided by millilitres. — using a standard filter from another spectrophotometer or haemoglobinometer for adjustment. e. together with a small volume of leukocytes. Note: If the spectrophotometer. It is of diagnostic value in patients suffering from anaemia. dehydration. the plasma volume fraction in the above example would be 550 ml/1000 ml = 0. haemoglobinometer or colorimeter was calibrated. shock or burns. which leads to concentration of blood cells.

the “packed cell volume” in the example given would be 45%. the erythrocyte volume fraction was called either the “haematocrit” or the “packed cell volume”.45 instead of 45%. but make them much lighter than the first set of lines. you can make one yourself using graph paper. you could use the one printed here for reading erythrocyte volume fractions.4. make a series of 10 marks at intervals of 4 mm. containing dried heparin (if capillary blood is used.28. the top sloping line will be marked 1. draw blood by pricking either: — the third or fourth finger (Fig. 9. make 10 marks at intervals of 6 mm. 9.29) — the lobe of the ear — the heel (infants) after sterilizing the chosen area with ethanol.3. On the right-hand vertical edge. Using a blood lancet.280 Manual of basic techniques for a health laboratory Before the introduction of SI units. This method is preferable to that using a macro scale: it is quicker. if venous blood mixed with EDTA dipotassium salt. Now. and it was reported as a percentage rather than a decimal fraction. Using a ruler. (Cover it with a sheet of plastic. marking each sloping line you have drawn as follows: 0. in the same manner. 15–20 cm wide. draw 10 sloping lines connecting each mark on the left margin to the corresponding mark on the right margin. Each light line should be drawn exactly in the middle of the space between each pair of heavy lines. 22) is used. 10% solution (reagent no.27) q q q Microhaematocrit centrifuge Scale reader (usually provided with the centrifuge) Capillary tubes. and blood from the finger can be used. following the printed lines of the graph paper. q q q q q Fig. starting at the bottom. again using a ruler. Instead of making your own scale. Materials and reagents (Fig. draw a second series of sloping lines..1 Micro-scale method Principle The blood (mixed with anticoagulant) is placed in a long capillary tube and centrifuged in a microhaematocrit centrifuge. 0. In the right margin. “heparinized” tubes are not required) Long fine capillary Pasteur pipettes (long enough to reach the bottom of the tubes) with rubber teat Filter-paper Soft wax or plastic modelling clay (or a Bunsen burner or spirit lamp) Sterile blood lancet 70% Ethanol. Continue up the left margin. 0. draw a series of heavy vertical lines at intervals of about 3 cm.) Method Collection of specimens Capillary blood specimens 1.1. in using SI units. In the left margin. but becomes 0. The level reached by the column of erythrocytes is read with a scale reader.2. write the same numbers opposite the other ends of the sloping lines. the numerical value does not change. In the traditional system. Your scale should look like the one in Fig. On the left-hand vertical edge.0. 9. 75 mm long with a 1. 9. Note that. ruled in millimetres. 9. .27 Materials for estimating the erythrocyte volume fraction using the micro scale If no scale reader is available. Finally. write “0”. etc.5-mm bore. opposite the bottom horizontal line of the graph paper.

Venous blood specimens 1. 9. Plug the other end of the tube (i. Check that it is completely plugged to a depth of about 2 mm. Fig. 9. 9. 9. It is useful to have ready a numbered stand containing plastic modelling clay. Fill about three-quarters of the tube. . the end that has not come into contact with the blood) with soft wax or plastic modelling clay (Fig.e. Apply the tip (circled with red) of a capillary tube containing dried heparin to the drop of blood (Fig.31). Haematology 281 Fig. seal the end of the tube by heating it carefully over a Bunsen burner or spirit lamp (Fig. Wipe away the first drop with filter-paper.30). 3. Collect a venous blood specimen as described in section 9.29 Taking a capillary blood sample The blood should flow freely or with very little pressure to the area.28 Micro scale for estimating the erythrocyte volume fraction Fig. The blood flows into the tube by capillarity.32). 9.9.2 and add it to a test-tube containing EDTA dipotassium salt solution.30 Technique for drawing blood into a capillary tube Alternatively. Leave it to cool in a horizontal position. so that each patient’s tube can be stuck upright next to the corresponding number. 9. 2.

The sealed end of the tube should point outwards. 9. Measurement technique 1. 9.34): — at the top.32 Sealing the capillary tube by flaming Fig. a very thin layer of leukocytes. Seal the tube as described in step 3 above. Using a capillary pipette. 9. Place the capillary tubes in the numbered slots in the centrifuge head. 3. Hold the tube against the scale so that the bottom of the column of erythrocytes (not the bottom of the tube) is aligned with the horizontal zero line (see Fig. a column of erythrocytes. 9. The erythrocyte volume fraction reading is made exactly at the top of the column of erythrocytes.282 Manual of basic techniques for a health laboratory Fig. 9. fill about three-quarters of a capillary tube with blood. a column of plasma. 9. — in the middle. 2. 4.31 Sealing the capillary tube with wax Fig. Check to make sure that the bottom of the column of . — at the bottom. After centrifugation.33 Placing the capillary tubes in a centrifuge 2.0 passes through the top of the plasma column. 3. Move the tube across the scale until the line marked 1. the tubes will show three layers (Fig. making sure that the number on the slot corresponds to the specimen number. away from the centre (Fig.33).35). Centrifuge at 3000g (for the period of time recommended by the manufacturer of the centrifuge — usually 10 minutes).

also check (by means of the heavy vertical lines) that the tube is vertical. but between a heavy line and a light line. For example. 9. L: leukocytes. 40%”. if the top of the column of erythrocytes is not on a line. Haematology 283 Fig.34 Centrifuged capillary blood sample E: erythrocytes. Note: If your laboratory has not yet changed to SI units and is still using the traditional system.9. P: plasma.05. Fig. its position can be estimated to the nearest 0. .4 in Fig.4” report the result as “packed cell volume.01. 0. the same chart can be used.35). Simply read the numbers as percentages instead of fractions. 5. The light intermediate lines represent intervals of 0. The line that passes through the top of the column of erythrocytes gives the erythrocyte volume fraction (0. instead of “erythrocyte volume fraction.35 Measuring the erythrocyte volume fraction using the micro scale erythrocytes is still on the zero line. 9. 9.

37–0.0. there is a linear relationship between the erythrocyte number concentration and the erythrocyte volume fraction. the erythrocyte volume fraction is roughly 0. High values High values are found in cases of loss of plasma.40–0. i. 0. but the formula for the calculation is slightly different: if C is the erythrocyte count. In terms of haemoglobin(Fe).4.37 (packed cell volumes of 40% and 37%. In men with anaemia the erythrocyte volume fraction is lower than 0. Relationship between the erythrocyte number concentration and the erythrocyte volume fraction Normally.003 = 0. dehydration (as in diarrhoeal diseases) and also (rarely) in polycythaemia. the erythrocyte volume fraction will normally be in the range (5 . If the erythrocyte number concentration is C ¥ 1012 per litre. the erythrocyte volume fraction will normally be in the range (C .4)/10. Relationship between the erythrocyte volume fraction and the haemoglobin concentration Normally. The erythrocyte volume fraction is about 0. there is a linear relationship between the erythrocyte volume fraction and the haemoglobin concentration.38–0. Example: A person with a haemoglobin concentration of 130 g/l will normally have an erythrocyte volume fraction of 130 ¥ 0. as a percentage. Low values Low values are found in patients suffering from anaemia.. the packed cell volume (haematocrit).39.6 Normal erythrocyte volume fractions and packed cell volumes. the relationship is similar.e. severe burns. by age group Age group Newborn infants Infants (3 months) Children (5 years) Women Men Erythrocyte volume fraction 0.003 times the haemoglobin concentration when the latter is expressed in grams per litre.43 0.2)/10 to (5 .4)/10.6 shows the reference ranges for different age groups. In traditional units.0.40 0.0.50 Packed cell volume (%) 50–58 35–40 38–44 37–43 40–50 Results Reference range Table 9.50–0. Example: If the erythrocyte number concentration is 5 ¥ 1012/l.58 0.2 to (C ¥ 10) .44 0. the .284 Manual of basic techniques for a health laboratory Table 9.0.46.48 to 0. will normally be in the range (C ¥ 10) . respectively).2)/10 to (C .4 and in women it is lower than 0. If the haemoglobin concentration is expressed in terms of millimoles of haemoglobin(Fe) per litre.35–0.05 times the figure for grams per litre.

determine the leukocyte number concentration (see section 9. q If the haemoglobin concentration is expressed in millimoles of haemoglobin(Fe) per litre: haemoglobin(Fe) = 9. Haematology 285 concentration is about 8. The layer will seem abnormally thick if the leukocyte number concentration is greater than 20 ¥ 109/l.6 mmol/l.4.6).9%).43 = 349 g/l (or 34.43 mean erythrocyte haemoglobin concentration = 150/0. Thus.6 mmol/l. Values below the lower limit of the reference range indicate that the erythrocytes are “hypochromic” (i.e. Example: q If the haemoglobin concentration is expressed in grams of haemoglobin per litre: haemoglobin concentration = 150 g/l erythrocyte volume fraction = 0.43 = 21. Therefore. Reference values Normally the mean erythrocyte haemoglobin concentration lies between the following limits: — lower limit: haemoglobin 322 g/l or haemoglobin(Fe) 20 mmol/l. erythrocytes are never “hyperchromic” (i. Haemoglobin forms about 95% of the erythrocyte mass. When the value falls within this range. — upper limit: haemoglobin 371 g/l or haemoglobin(Fe) 23 mmol/l.9. It is normally very thin.e. Mean erythrocyte haemoglobin concentration The mean erythrocyte haemoglobin concentration is a measure of the average haemoglobin content of the erythrocytes. but they may increase in volume and thus be capable of containing more haemoglobin than normal. the mean erythrocyte haemoglobin concentration should be determined again. if it seems thick. 349 g/l ¥ 0. Note: to convert values in g/l to values in mmol/l. 9.34).43 mean erythrocyte haemoglobin concentration = 9. when the leukocyte number concentration may be 100–200 ¥ 109/l.0 ¥ 0.05 = 0. Additional information provided by the erythrocyte volume fraction test Examine the layer of leukocytes just above the column of erythrocytes (see Fig. of normal colour).062 06 = 21.3 mmol/l erythrocyte volume fraction = 0. If the value is higher than the upper limit of the reference range. .3/0. multiply by 0. in this case the mean 1 See note about expression of haemoglobin concentration on page 284. using the above example.e. the erythrocytes are said to be “normochromic” (i. In cases of leukaemia. less coloured than normal). and the erythrocyte volume fraction will be about 8. the layer may be several millimetres thick. more coloured than normal).062 06.0 mmol/l. It is expressed either in grams of haemoglobin per litre or in millimoles of haemoglobin(Fe) per litre1 and is calculated by dividing the haemoglobin concentration of the blood by the erythrocyte volume fraction. Low values are found in patients with hypochromic anaemia.

Using the capillary pipette. 9. Materials and reagents (Fig. but it never exceeds these values. 22) or Wintrobe solution (reagent no. 9. 2.6 mmol/l).36 Principle of the macro scale for estimating the erythrocyte volume fraction 1. 9. 65).38 Using a capillary pipette to fill a graduated tube with blood . fill the graduated tube with blood up to the 100 mark.0/43) ¥ 100 = 35%. The mean erythrocyte haemoglobin concentration is usually called the “mean corpuscular haemoglobin concentration” (MCHC).36). It can also be expressed as a percentage. 9. This is calculated by dividing the haemoglobin concentration of the blood in grams per 100 ml by the packed cell volume as a percentage and multiplying by 100.5 cm long with a 0. q q Method Collection of specimens Fig. Example: haemoglobin concentration = 15. the reference range is 32–37%. Collect a venous blood specimen as described in section 9. 9. calibrated from 0 to 100 Long fine capillary Pasteur pipette (long enough to reach the bottom of the tube) with rubber teat Anticoagulant — EDTA dipotassium salt. In this system.38). making sure that no air bubbles form (Fig. 9. the test should be repeated.0 g/100 ml packed cell volume = 43% MCHC = (15. 9.2 Macro-scale method Principle The blood (mixed with anticoagulant) is placed in a graduated tube and centrifuged to pack the erythrocytes. The level of the column of erythrocytes is then read directly in the graduated tube (Fig.286 Manual of basic techniques for a health laboratory erythrocyte haemoglobin concentration may be as high as 380 g/l (haemoglobin(Fe) 23. Fig. 10% solution (reagent no. If such a result is obtained.37) q q Centrifuge Special graduated tubes (Wintrobe tubes).2 and add it to a graduated tube containing anticoagulant (see above).6-cm bore.37 Materials for estimating the erythrocyte volume fraction using the macro scale Fig. The MCHC never exceeds 38%.4. 9.

with a 20-cm arm 3100 rpm will be needed.4 ¥ 106 4. Reference range Table 9.2 ¥ 106 4. Place the graduated tubes in the centrifuge and centrifuge for 30 minutes at 2300g. 9. 9. Low values Patients with anaemia caused by insufficient production. 3600 rpm will be needed to attain this force.39).7 Normal erythrocyte number concentrations.0 ¥ 1012 3. Unfortunately.5–6.5 Estimation of the erythrocyte number concentration The number of erythrocytes contained in 1 litre of blood is called the erythrocyte number concentration.9. Results See page 282. by age group Age group Erythrocyte number concentration SI units (per litre) Newborn infants Infants (1–6 months) Children (4 years) Women Men 5.7 shows the reference ranges for different age groups. The figure obtained is a percentage (the “packed cell volume”). divide by 100 to obtain the erythrocyte volume fraction. Important: a force of less than about 2300g will give a false result.39 Measuring the packed cell volume 9. such instruments are often not available in peripheral laboratories.5–6. The Table 9.4) or the haemoglobin concentration (see section 9.8–5.4 ¥ 106 4. loss or haemolysis of erythrocytes will have low erythrocyte number concentrations.4 ¥ 10 12 12 12 Traditional units (per mm3) 5.8–5.) Accurate methods for counting erythrocytes require an electronic counter system.0–7.0–7. A simple but far less accurate method uses a counting chamber in which erythrocytes are counted under the microscope.0–5. it is expressed as the number of erythrocytes per cubic millimetre and is called the erythrocyte or red cell “count”.3) is measured and the erythrocyte number concentration calculated.9 ¥ 10 3.8–5.9 ¥ 106 3. It is recommended instead that the erythrocyte volume fraction (see section 9. Make sure that the correct set of graduations is being used.0 ¥ 106 3. 2. Fig. upwards towards the 100 mark.8–5. However. Haematology 287 Measurement technique 1. If the rotor arm of the centrifuge (measured from the axis of rotation to the base of the bucket holding the tube) is 15 cm long. High values Patients who are dehydrated or have polycythaemia will have high erythrocyte number concentrations. The clinical picture is determined by the extent and duration of anaemia. Read the level at which the erythrocytes meet the layer of leukocytes (Fig.0–5. Note: Anaemia is a clinical syndrome that has many different underlying causes. this method is of such low precision that it should not be used. (In traditional units.2 ¥ 1012 .4 ¥ 10 4.

9. and the number of cells per litre of blood is calculated. Other causes of anaemia are: — trauma — parasitic infections — diseases of the endocrine system — chronic diseases — inborn errors of metabolism — intoxication.6 Estimation of the leukocyte number concentration The number of leukocytes contained in 1 litre of blood is called the leukocyte number concentration or leukocyte count. dizziness and irritability to uncontrolled behaviour and even shock and cardiac insufficiency. malnutrition and vitamin deficiencies usually contribute to anaemia in association with iron deficiency. whereas in typhoid fever there is a marked decrease. graduated to the 50-ml (0. Anaemia may result from: — blood loss — decreased production of erythrocytes — increased destruction of erythrocytes.40 Neubauer counting chamber .05-ml) mark q Fig. 9. but leaves the leukocytes intact.288 Manual of basic techniques for a health laboratory symptoms range from pallor and mild fatigue to headache.1 Principle The blood is diluted in a leukocyte diluting fluid which haemolyses the erythrocytes.6.6. Other common causes such as infection.2 Materials and reagents q q Microscope Ruled counting chamber — preferably the improved Neubauer chamber (Fig.40. 9. For example. The leukocytes are then counted in a counting chamber under the microscope. The most common cause of anaemia worldwide is iron deficiency. 9. the Bürker chamber is rarely used) Blood (Sahli) pipette. in infectious mononucleosis and bacterial infections there is a marked increase. In certain diseases the number of leukocytes in the blood is altered. 9. malaria.

Take care not to overfill beyond the ruled area. — depth = 0. Label the bottle with the patient’s name and/or number. Do not allow air bubbles to enter. Draw venous or capillary blood to the 0. Rinse the pipette by drawing in and discharging fluid from the bottle three times. coloured bands called Newton’s rings appear between the two glass surfaces. 9. 3. 2. Mix the diluted blood well.3 Method 1. Haematology 289 q q q q Graduated pipette. Wipe the outside of the pipette with absorbent paper. pressing it carefully into place. and making up the volume to 100 ml with distilled water). With venous blood ensure that it is well mixed by inverting the bottle containing it and the anticoagulant repeatedly for about 1 minute immediately before pipetting it. Attach the coverslip (supplied with the chamber) to the counting chamber.6.05-ml mark (Fig.95 ml of diluting fluid into a small bottle using the 1-ml graduated pipette.42). check that the blood is still on the 0.41).1 mm. 5. The dimensions of the Neubauer ruled chamber are as follows: 9. Use a Pasteur pipette or a capillary tube to fill the counting chamber (Fig. 1 ml Pasteur pipette or capillary tube Hand tally counter or bead counter Diluting fluid (prepared by adding 2 ml of glacial acetic acid to 1 ml of gentian violet. 9. Fig. 1% aqueous solution. The dilution of the blood is 1 in 20. 9. and expel it into the bottle of diluting fluid.05-ml mark of the blood (Sahli) pipette. Pipette 0. When the coverslip is properly attached. — area = 9 mm2. 4.41 Checking that the blood is still on the mark .9.

9. 6.44 Counting leukocytes using the improved Neubauer ruled chamber . Example: number of leukocytes counted = 188 number of leukocytes per litre = (188 ¥ 0.1 mm. Include in the count the leukocytes seen on the lines of two sides of each square counted. Place the chamber on the stage of the microscope.1 = 0. 9. 8. Count the leukocytes in an area of 4 mm3 of the chamber. Focus the rulings of the chamber and the leukocytes. as shown in Fig. 7 and 9 as shown in Fig. and refill with another drop. This square represents one of the four counted. Use the ¥ 10 objective and the ¥ 10 eyepiece. 9. 9.05) ¥ 109 Result reported: 9.4 mm3. Reduce the amount of light entering the condenser by adjusting the iris diaphragm. therefore the volume in which leukocytes are counted is 4 ¥ 0. 9. Leave the counting chamber on the bench for 3 minutes to allow the cells to settle. The chamber depth is 0. Finally. Since the dilution is 1 in 20.290 Manual of basic techniques for a health laboratory Fig. 3. 7. the total area is therefore 4 mm2. Thus division by four and multiplication by 10 will give the number of leukocytes in 1 mm3 of diluted blood.43. Calculate the number of leukocytes in 1 litre of blood by multiplying the number of leukocytes counted in the four squares by 0. Do not mistake pieces of dust for leukocytes. multiplication by 20 will give the number of leukocytes in 1 mm3 of undiluted blood. 9. you must start again: remove and clean the coverslip.05.42 Filling the counting chamber Fig.4 ¥ 109/l Explanation of calculation Each of the four squares in which leukocytes are counted has an area of 1 mm2. clean the counting chamber. using the corner squares numbered 1.43 Using the improved Neubauer ruled chamber Important: If the liquid overflows into the channel between the two chambers. there are 1 million (106) cubic millimetres in Fig.44. Report the result as “number by 109/l)”.

Haematology 291 1 litre.05 ml of blood and 0. In leukaemia. This can occur with certain bacterial infections. multiply by 100 instead of 50 to give the number per cubic millimetre.05 ml of blood and 1. (If traditional units are being used.05 ¥ 109 Example: A total of 188 leukocytes are counted in the four squares. Table 9. In this case it is necessary. which gives a dilution of 1 in 40. the number of cells counted is multiplied by 0. the number of cells counted is multiplied by 0.8. The number of leukocytes per cubic millimetre of undiluted blood is therefore: 188 ¥ 10 ¥ 20 (= 188 ¥ 50 = 9400) 4 and the number per litre is: 9400 ¥ 106 = 9.8 Normal leukocyte number concentrations.4 Results Reference range The reference ranges for different age groups are given in Table 9. when determining the number concentration. Leukopenia is also seen following treatment with certain drugs. by age group Age group Newborn infants Infants (3–9 months) Children (3 years) Children (10 years) Adults a Leukocyte number concentration (per litre)a 10–20 ¥ 109 4–15 ¥ 109 4–11 ¥ 109 4–10 ¥ 109 4–10 ¥ 109 The reference range may be different in certain indigenous populations.05 to give the number ¥ 109 per litre.45 ml of diluting fluid. When the leukocyte number concentration is very low. it is necessary to dilute the blood less — for example 0. leukocyte number concentrations of 50 ¥ 109/l to 400 ¥ 109/l. . or even higher values.1 instead of by 0.05 in order to give the number ¥ 109 per litre.6. High values An increase in the total number of circulating leukocytes is called leukocytosis.95 ml of diluting fluid. This can occur with certain infections including typhoid fever and malaria. can be found. If this dilution is used.25 instead of by 0.4 ¥ 109 9. This can be summarized as follows: number of leukocytes per litre = leukocytes counted ¥ 10 ¥ 20 ¥ 106 4 = leukocytes counted ¥ 50 ¥ 106 = leukocytes counted ¥ 0. which gives a dilution of 1 in 10. so multiplication by 106 will give the number of leukocytes per litre of undiluted blood.9. If this dilution is used.) Low values A decrease in the total number of circulating leukocytes is called leukopenia. to use a greater dilution of blood — for example 0.

90) are early stages of erythrocytes. 60) (keep in refrigerator) or EDTA dipotassium salt. When normoblasts are present in large numbers and the leukocytes are counted using a fully or semi-automated cell counter.5 mm. Collect 2 ml of blood into a syringe. The height of the column of plasma after 1 hour indicates the sedimentation rate of the erythrocytes (erythrocyte sedimentation rate (ESR)).) Westergren stand Test-tubes Graduated syringe. 22).7.7 Measurement of the erythrocyte sedimentation rate 9. 9.4 ml of trisodium citrate solution into a test-tube or bottle.10) and count the number of normoblasts seen for every 100 leukocytes. 9.2 Materials and reagents (Fig.7. Examine a thin Romanowsky-stained blood film (see section 9. They are not normally present in the blood. 2. . the number concentration of normoblasts is: 50 ¥ 16 = 5. the leukocyte number concentration must be corrected as follows.45) q Westergren ESR tube: internal diameter 2. 5 ml Timer Anticoagulant: trisodium citrate. Apply the tourniquet as loosely as possible. 5 ml Graduated pipette. The erythrocytes settle to the bottom. Collect a venous blood specimen (see section 9.2% solution (reagent no.3 Method 1. Normoblasts are not haemolysed in the diluting fluid and are therefore counted with the leukocytes.7. 2 to 20 mm.1 Principle Blood collected into an anticoagulant is placed in a long graduated tube held in a vertical position. leaving a layer of plasma above. Pipette 0. etc.5. graduated from 0 to 200 mm (often marked 1 to 20: 1 corresponds to 10 mm. 9.7 ¥ 109/l. puncture the vein at once and release the tourniquet.2). Calculation: The number concentration of normoblasts (per litre) is: number of normoblasts counted ¥ leukocyte number concentration 100 + number of normoblasts counted Example: If 50 normoblasts are counted and the leukocyte number concentration is 16 ¥ 109/l. but they may be present in the blood in certain diseases such as sickle cell anaemia or other haemolytic anaemias.3) ¥ 109/l = 10. 9.3 ¥ 109 l 100 + 50 and the corrected leukocyte number concentration is: (16 . q q q q q q 9.292 Manual of basic techniques for a health laboratory Correction for nucleated erythrocytes Nucleated erythrocytes or normoblasts (see Fig. 10% solution (reagent no. 3.

Measurement of the ESR should begin within 2 hours of collection of the blood. Check that there are no air bubbles in the tube. 9. . 6.47). 9. 5. Remove the needle from the syringe and add 1. Draw the citrated blood into the Westergren tube (using a rubber safety bulb) up to the 0-mm mark (Fig.49). These include acute and chronic infections. 9. Note: If a patient is dehydrated measurement of the ESR has little value. free from draughts.45 Materials for measuring the erythrocyte sedimentation rate Fig. 9. myocardial infarctions and rheumatoid arthritis.9 shows the reference ranges for adults.46).0 ml) (Fig. Check that the stand is level.48). 4.4 Results The result is expressed in millimetres per hour (mm/h). 9. not close to a central heating radiator and not in direct sunlight. making sure that the tube is completely upright (Fig.46 Adding a blood sample to the trisodium citrate solution 3. 7. Leave on a bench away from vibration (e. Place the tube in the Westergren stand. not on the same bench as a centrifuge). High values Any disease that produces plasma protein changes will increase the ESR. 9. Reference range Table 9.7. Wait 1 hour (set the timer to ring).9.g.6 ml of blood to the test-tube containing anticoagulant (marked to contain a total of 2. The ESR is also increased in patients suffering from anaemia (see page 285). Haematology 293 Fig. Shake gently. then note the height of the column of plasma in mm graduations starting from the 0-mm mark at the top of the tube (Fig. 9.

a by age group Age group Adults (< 50 years) Men Women Adults (> 50 years) Men Women a ESR (mm/hour) < 15 < 20 ≥ 20 ≥ 30 At an ambient temperature of 25 °C.9 Erythrocyte sedimentation rates (ESR). Fig.294 Manual of basic techniques for a health laboratory Table 9.49 Measuring the height of the plasma column . 9. 9. 9.47 Drawing citrated blood up to the 0-mm mark of the Westergren tube Fig.48 Placing the Westergren tube in the stand Fig.

8. without any need to squeeze the ear lobe.52).51 Puncturing the ear lobe .8 Measurement of the bleeding time: Duke method 9.9. 4. a little further along the strip of paper (Fig.8. 9. Puncture the ear lobe (Fig. The blood should flow freely. 9. Gently clean the lobe of the ear with cotton wool and ether (Fig. 9. Start the stopwatch. 9. 9. 9.50).1 Principle A small cut is made with a lancet in the lobe of the ear. if available. 9.2 Materials and reagents q q q q q Sterile blood lancets Microscope slides Filter-paper (or blotting paper) Stopwatch. The test is performed: — for the diagnosis of certain haemorrhagic disorders. Do not rub. — before surgical operations.53).50 Cleaning the ear lobe with ether Fig. Do not touch the skin with the paper. After 30 seconds collect the first drop of blood on a corner of the filter-paper (or blotting paper) (Fig. 9. Blood flows from the puncture and the time it takes for the bleeding to stop is measured. 9. 2.51). — before puncture of the liver or spleen. otherwise a watch with a second hand Ether. 3. Wait 30 seconds more. trypanosomiasis and malignant diseases. Haematology 295 Very high ESR values occur in tuberculosis. Allow to dry. Fig. The ESR is also raised in pregnancy.8. Collect the second drop of blood in the same way.3 Method 1.

9. 9.8. 9. collect the first drop of blood Fig. Continue to collect one more drop of blood every 30 seconds.53 After 1 minute.4 Results Report the bleeding time to the nearest half minute. 9. 9. collect the second drop of blood Fig.55 Calculating the bleeding time . Fig. Another method is to count the number of drops on the filter-paper (or blotting paper) and multiply by 30 seconds (Fig. 9.296 Manual of basic techniques for a health laboratory 5.54). examine a Romanowsky-stained thin blood film (see section 9. For example: there are seven drops. The bleeding time is 7 ¥ 30 seconds = 3. 6. Duke method: 1–5 minutes). Mention also the reference range for the method used. Example: bleeding time 3.10) to see whether the number of thrombocytes appears to be less than normal (venous blood must be used).54 Continue to collect one more drop of blood every 30 seconds Fig.5 minutes. stop the stopwatch (or note the time on the watch).55). The drops become progressively smaller (Fig.5 minutes (normal range. 9. If the bleeding time is prolonged. When no more blood appears.52 After 30 seconds.

9. 2. These tests are carried out for the diagnosis of certain haemorrhagic disorders.9. 9.9.56). 9. 48 and 72 hours until lysis occurs. Fig. although it begins to retract. 9. until the clot dissolves completely and all the erythrocytes sink to the bottom of the tube (Fig.1 Principle The tubes with clotted blood are used: — for observation of the retraction of the clot — for measurement of the time it takes for the clot to dissolve (lysis). 24.9 Observation of clot retraction and measurement of lysis time 9. the red cell mass separating from the yellow serum (Fig. The clot normally remains solid during the first 4 hours. Do not add anticoagulant to the tubes in which you collect the blood. 3 and 4 hours. usually in the first hour. Observation of clot retraction 1. Measurement of lysis time 1. 75 mm ¥ 10 mm. marked to hold 1 ml Timer Metal test-tube rack Water-bath Materials to carry out venepuncture (see section 9. After 4 hours the clot will have completely retracted.9.2). Haematology 297 9.57).2 Materials q q q q q Glass test-tubes. Place the tube in the water-bath at 23°C (or leave it to stand at room temperature).9. 2. 9.56 After 4 hours.2.3 Method Collection of specimens Collect a venous blood specimen from patients as described in section 9. Examine the clot after 1. 2.2. Place the tube containing the blood in the water-bath at 37°C (or leave it to stand at room temperature). examine the clot . that is. Examine the clot after 12.

58 Normal clot retraction (Examine a Romanowsky-stained thin blood film.62). using venous blood. Lysis time A clot normally takes at least 48 hours to dissolve.) Abnormal plasma proteins Abnormal plasma proteins cause coagulation of plasma.60). 9. Fig.60 Blood deficient in thrombocytes Fig. 9. However.58). Haemophilia is a hereditary haemorrhagic disease affecting males. Hardly any serum will have exuded.57 Lysis Blood deficient in fibrinogen If blood is deficient in fibrinogen there is a small red clot at the bottom of the tube. 9.59 Blood deficient in fibrinogen Fig.62 Haemophilia . not necessarily attached to the sides of the tube. at the surface.9.298 Manual of basic techniques for a health laboratory 9. For example.61). 9. Abnormal clot retraction Fig. 9. Beneath it is a poorly retracted red clot (Fig. Blood deficient in thrombocytes If blood is deficient in thrombocytes there is a red clot that remains almost completely attached to the sides of the tube and that will have retracted very little.4 Results Normal clot retraction The red clot is well separated and. It is surrounded by sedimented erythrocytes and covered by supernatant (Fig.10. is attached to the sides of the tube (Fig. 9. Haemophilia If there is no clot at all or a yellow clot that forms very slowly over the deposit of erythrocytes (Fig. This appears as a yellow clot — clotted plasma. 9. in patients with acute fibrinolytic disease the clot may dissolve within 1–4 hours. Report the lysis time of the clot in hours. the lysis time may be reduced in certain conditions. with a description of the clot. 9. see section 9. Fig. if at all (Fig. 9.61 Blood containing abnormal plasma proteins Fig. Report the clot retraction as: — “normal” — “abnormal”. 9. There may be a small deposit of erythrocytes in the bottom of the tube. 9. the cause is a serious clotting factor deficiency such as that which occurs in haemophilia.59). it should not be more than 5 mm thick.

50 ml or 100 ml Beakers or bottles containing clean tap water Wash bottle containing buffered water (reagent no. which contain essential azure B and eosin dyes. then staining with pre-prepared diluted stains. Haematology 299 9. cleaned with ethanol or ether using a piece of soft cloth (Fig.14).4) — identifying certain parasites (see section 4. Field stains A and B. 38) EDTA dipotassium salt. q q q q q q q q q q q q q q q q . 10% solution (reagent no. Thin blood films are stained with Romanowsky stains.9.10.10 Preparation and staining of thin blood films 9. either over a sink or over a staining tank Measuring cylinder. Better results are obtained by fixing first with methanol.10. if necessary. After staining. Field stains are used for both thin and thick blood films. which is used with Giemsa stain. 22) Methanol 70% Ethanol or ether. 15) Interval timer Rack for drying slides Field stain (reagent no. 9.1 Principle A thin blood film is prepared by spreading a small drop of blood evenly on a slide so that there is only one layer of cells. 34) May–Grünwald stain (reagent no.13) — detecting abnormal erythrocytes (see section 9. May–Grünwald stain. which can be used alone or together with May–Grünwald or Jenner stain. The Romanowsky stains most widely used include: q q q Leishman stain. which are prepared in water unlike the above-mentioned stains. Giemsa stain. 25) Giemsa stain (reagent no. which are made up in methanol. 9.63)) Spirit lamp or Bunsen burner Glass spreader (see below) Blood lancet Two glass rods. 29) Leishman stain (reagent no. blood films are used for: — determining leukocyte type number fractions (see section 9.7) — estimating the number of thrombocytes (see section 9. which is used alone. q The Romanowsky stains prepared in methanol can be used to fix thin films before being diluted on the slides to stain the films. as described below.10.2 Materials and reagents q q Microscope Microscope slides (should be well washed and.

9. First take samples for determining the erythrocyte or leukocyte number concentrations. Hold the slide with one hand. Let the blood flow freely. 2. if possible (see sections 9.3 Method Collection of specimens Take the blood from the side of the third or fourth finger as shown in Fig. 9. Make a diagonal mark across the two corners at one end of the slide with a file. 9.5 and 9. . Preparation of the film 1. Important: Do not take blood from: — the index finger or thumb — an infected finger (e.29. Collect a drop of blood of about 4 mm diameter by touching it lightly with one end of the slide (Fig. 9.65).64). EDTA dipotassium salt solution should be added. Using the other hand. If it is not possible to prepare the film within 1–2 hours of collection of the blood specimen. 9. Snap off the two filed corners (Fig.g. place the edge of the spreader just in front of the drop of blood (Fig. paronychia) — the ear (too many monocytes). 9.66).300 Manual of basic techniques for a health laboratory Fig. Other anticoagulants such as heparin alter the appearance of leukocytes and thrombocytes and should not be used.10.63 Cleaning slides to be used for thin blood films To make a spreader. select a slide with a perfectly smooth edge.6).

9.68). Let the blood run along the edge of the spreader (Fig. 4.69) (all the blood should be used up before you reach the end). Draw the spreader back until it touches the drop of blood (Fig. 9. Haematology 301 Fig.9.64 Making a glass spreader Fig. . 9. Push the spreader to the end of the slide with a smooth movement (Fig 9.66 Position the spreader in front of the drop of blood 3. Blood from patients with anaemia should be spread more rapidly.65 Collecting a drop of blood on a slide Fig. 9. 9.67). 5.

9. 9. A well-spread film is of great importance.70 (b). q The film must not contain holes because a greasy slide has been used. not ragged and lined as shown in Fig. q The film must not be too long.70 Correctly (a) and incorrectly prepared (b) blood films 6.302 Manual of basic techniques for a health laboratory Fig.69 Push the spreader to the end of the slide with a smooth movement Fig. A badly spread film will give the wrong leukocyte type number fractions and make it impossible to report erythrocyte morphology. q There should be no lines extending across or down through the film. Check that the film is satisfactory as shown in Fig. q The film must be smooth at the end. 9. 9.68 Allow the blood to run along the edge of the spreader Fig.70 (a). .67 Draw the spreader back until it touches the drop of blood Fig. 9. q The film must not be too thick. 9.

A number of precautions are also required to avoid staining the films too blue. Prepare sufficient stain for 1 day’s use only. Haematology 303 Drying the film Adequate drying is essential to preserve the quality of the film. In the wet season (in the tropics) Dry the film by waving it rapidly about 5 cm away from the flame of a spirit lamp or Bunsen burner: hold the slide to the side and slightly above (but never directly over) the flame (Fig.4. Mix. alkaline water one that is too blue. these are briefly described below. The preparation technique is described in section 2.71 Drying the blood film over a spirit lamp Fixation of the film If the film is intended for determining leukocyte type number fractions. q Staining of the film Method for Leishman stain 1. It should be between 6. If necessary. except for Field stain). protect the blood film from flies. Important: The staining time may need to be adjusted.4. Do not tip the stain off as this will leave a deposit of stain on the film. Use neutral water (buffered if possible.2. especially in humid climates. Remove stain deposits with methanol. Wash the stain off in a stream of buffered water. Tip the water off and place the slide in a draining rack to dry. Acid water produces a film that is too red. Fig.8 and 7. it should be fixed with methanol before staining with May–Grünwald stain (see below). The film can be left to air-dry in dry climates. Do not use acid. (The time taken for differentiation will depend on the stain and the pH of the water used. If the film is intended for detection of parasites. Prepare a 1 in 3 dilution of Leishman stain using one volume of stain and two volumes of buffered water. Example: Use 10 ml of stain and 20 ml of buffered water. Wash it every day. 2. too pink or too dark.2. 4. 5. 9. . it should be fixed with methanol before staining with Giemsa or Field stain (see below). Precautions Care is required to avoid the formation of deposits of stain. and preferably between 7. especially when a new batch of stain is received or the stain has been stored for a long time. Neutral water must be freshly prepared as it becomes acid when exposed to air. Cover the slide with the diluted stain for 7–10 minutes. which appear on the film as masses of little black spots. q Use perfectly clean glassware.71). 9. 3.0 and 7. as the diluted stain does not keep well. Mark the dry film with the patient’s name or number.9. 6. Write with a lead pencil on the thick part of the film not used for examination. Leave clean water on the slide for 2–3 minutes to differentiate the film.) The pH of the water is of vital importance in differentiating the different types of leukocyte with Leishman stain. Fix the thin blood film with methanol for 2–3 minutes.

The time for differentiation will depend on the stain and the pH of the water used. q Dilute Giemsa stain 1 in 10 using one volume of stain and nine volumes of buffered water.72) and count up to five. Wash the stain off in a stream of buffered water. Cover the slide with diluted May–Grünwald stain for 5 minutes. 5. Example: use 2 ml of stain and 18 ml of buffered water. Fix the thin film with methanol for 2–3 minutes. Dip the slide into Field stain B (Fig.73 Rinsing the slide in water . as the diluted stains do not keep well. 2. Tip the stain off and replace with diluted Giemsa stain for 10 minutes. 6.8 and 7. Drain and wash the slide well in the second container of tap water.0. 9. The pH should be between pH 6. Prepare the Giemsa mixture slowly and carefully. Method (rapid) for Field stain 1.72 Staining the blood film with Field stain Fig. especially when a new batch of stain is received or the stain has been stored for a long time. 9. Tip the water off and place the slide in a draining rack to dry. 9. Example: use 10 ml of stain and 10 ml of buffered water. Note: Prepare only enough stain for 1 day’s use. Fix the thin film with methanol for 2–3 minutes. Important: The staining time may need to be adjusted. Prepare the stains as follows: q Dilute May–Grünwald stain 1 in 2 using equal volumes of stain and buffered water. 4. Drain and dip the slide into Field stain A and count up to 10. 7. Drain and wash the slide in the first container of tap water (Fig. 3. 2. 3. Fig.304 Manual of basic techniques for a health laboratory Method for May–Grünwald and Giemsa stains 1. 9. Shaking causes the stain to precipitate. Mix gently. Leave clean water on the slide for 2–3 minutes to differentiate the film.73). Do not tip the stain off as this will leave a deposit of stain on the film. Mix.

74). Basophilic staining can usually be prevented by using buffered water at a more acid pH and. neither too blue nor too pink. Rinse with methanol. It should appear mauve. but not overlapping (Fig. Wash at once in neutral water.10 Appearance of blood cells in thin films stained with Leishman stain Cell type Neutrophils Eosinophils Basophils Monocytes Lymphocytes large small Erythrocytes Thrombocytes Cytoplasm stains clear blue Cytoplasm stains dark blue Stain pink-red Stain mauve-pink Appearance Cytoplasm stains faint pink and contains small mauve granules Cytoplasm stains faint pink and contains large red granules Cytoplasm contains numerous dark mauve-blue granules Cytoplasm stains grey-blue . where the cells are too closely packed (Fig. look at the cells just before the thin end of the film. size or colour. as needed.76). examine the slides. 9. Do not look at either the thick end. Rinse the slide twice in methanol. if necessary. return the slide either to the Field stain A or to the Field stain B for a few more seconds. The cells should appear as described in Table 9. The various types of abnormal erythrocytes are described below. Erythrocytes In certain diseases.4 Microscopic examination Using the ¥ 40 objective. Too much blue in the film (basophilic staining) Prepare a solution of 1% boric acid in 95% ethanol. where there are not enough cells (Fig. How to remedy poor results Deposits of May–Grünwald stain or neutral water Deposits caused by May–Grünwald stain or neutral water can be seen with the naked eye in the liquid on the slide.10. To check for abnormal erythrocytes.10. especially anaemia. Dry and examine under the microscope without further treatment.9. If the film is not satisfactory. Dry and re-stain using fresh or filtered May–Grünwald stain. If the film is satisfactory. Drain off the stain. or the thin end. stand the slide in a draining rack to dry. but wash off immediately with neutral water. Examine the colour of the film. the erythrocytes may have an abnormal shape. Table 9. just touching one another. altering the differentiation time.75). 9. this is where they are spread out. Rinse the slide twice in this preparation. Dry the slide and repeat the staining procedure from the beginning. 9. Deposits of Giemsa stain These deposits are seen with the naked eye or under the microscope. Poor staining may also be caused by impurities in the dyes — the use of the standardized stain is recommended. Haematology 305 4. 9.

vitamin B6 deficiency. discoid. but between them there is a colourless ring. Target cells (Fig.78) Size: 6–8 mm.75 Examining blood films for abnormal erythrocytes: cells too closely packed Fig. sickle-cell anaemia and iron-deficiency anaemia. Shape: round or slightly irregular. centre pale pink or colourless. Cytoplasm: periphery deep pink.77) Size: 6–8 mm. 9. liver diseases. . Cytoplasm: centre and periphery stain well.76 Examining blood films for abnormal erythrocytes: not enough cells Normal erythrocytes (Fig. 9.74 Examining blood films for abnormal erythrocytes: where to look Fig. Seen in thalassaemias. occasionally slightly irregular.306 Manual of basic techniques for a health laboratory Fig. 9. 9. haemoglobinopathy. Shape: round. 9.

9. 9. Microcytes (Fig. Must be distinguished from spherocytes (see below). 9. Macrocytes (Fig. 9.80 Microcytes Sickle cells (Fig.78 Target cells Fig. vitamin B12 deficiency and iron-deficiency anaemia.81) Size: large (9–10 mm). Haematology 307 Fig. target cells and often macrocytes.4. 9. sideroblastic anaemia and thalassaemias. 9. Seen often in iron-deficiency anaemia.77 Normal erythrocytes Fig. and in certain liver diseases.11. Seen in sickle-cell anaemia and sickle-cell thalassaemia. often with one or both ends curved and pointed.9. Must be distinguished from reticulocytes (see below). Seen in macrocytic anaemias caused by folic acid deficiency. Microscopic examination of sickle cells in wet preparations is described in section 9.80) Size: small (about 5 mm). .79) Shape: elongated and narrow. 9.79 Sickle cells Fig. along with nucleated erythrocytes.

Shape: perfectly round.82 Schistocytes Fig.84 Elliptocytes Schistocytes (Fig.308 Manual of basic techniques for a health laboratory Fig. Seen in haemolytic anaemias and hereditary spherocytosis. Seen in haemolytic anaemias. Cytoplasm: stained darker at the periphery (especially at the poles). 9. 9. .83) Size: small (6 mm). Shape: oval. 9. 9.83 Spherocytes Fig.81 Macrocytes Fig. sickle-cell disease and thalassaemias. 9. 9. Cytoplasm: more darkly stained than normal erythrocytes.84) Size: normal (8 mm).82) Size: normal or slightly smaller than normal erythrocytes. 9. Elliptocytes (Fig. Spherocytes (Fig. Fragmented cells.

triangular. Do not confuse with stain deposits. 9. Seen in many types of severe anaemia and myelofibrosis. Seen in severe anaemias. Erythrocytes containing basophilic stippling (Fig. iron-deficiency anaemia. oval.85) A condition in which erythrocytes of different sizes are present in the blood. Poikilocytes (Fig. Erythrocytes containing Cabot ring bodies (Fig. e. pernicious anaemia. Do not confuse with malaria parasites. 9. Found in hereditary elliptocytosis. and following splenectomy. 9.9. 9. Seen in haemolytic anaemias and megaloblastic anaemia. Haematology 309 Fig.89) Erythrocytes containing multiple blue–black dots in the cytoplasm. 9.87 Erythrocytes containing Howell– Jolly bodies . Seen in vitamin deficiency.87) Erythrocytes containing one or more large purple granules (nuclear remnants). 9. Do not confuse with thrombocytes lying on the cells. Fig. pear-shaped and indented cells.88) Erythrocytes containing thin ring-shaped or figure-of-eight structures that stain red with Wright stain. Seen in many types of anaemia. erythrocytes measuring 9 mm are mixed with erythrocytes measuring 6 mm. Anisocytosis (Fig. a mixture of round.85 Anisocytosis Fig. e. thalassaemias and lead poisoning. Erythrocytes containing Howell–Jolly bodies (Fig.g.86) Erythrocytes of different shapes in the blood.g. sickle-cell disease.86 Poikilocytes Seen very occasionally. 9. 9. thalassaemias and myelofibrosis.

9. Shape: rounded. As already mentioned. for example sickle-cell anaemia. with deep purple. known as the leukocyte type number fraction. 9. dense chromatin. often eccentric. 9. 9. eosinophils. there are five main types of leukocyte — neutrophils. Nucleus: round. and in leukaemias and cancers. Fig. . basophils.90) Size: 8–10 mm.90 Normoblasts Leukocytes In contrast to erythrocytes.88 Erythrocytes containing Cabot ring bodies Fig. The proportion of each type of leukocyte.92) Size: 12–15 mm.310 Manual of basic techniques for a health laboratory Fig. eosinophils and basophils) Polymorphonuclear cells have: — a nucleus with several lobes. Seen in accelerated erythropoiesis in severe anaemias. well defined. Polymorphonuclear cells (neutrophils. lymphocytes and monocytes. in severe bacterial infections. 9. Cytoplasm: pink or greyish-blue. 9. leukocytes contain a nucleus that may vary in size and shape.89 Erythrocytes containing basophilic stippling Nucleated erythrocytes (normoblasts) (Fig. Shape: round or irregular. — granules in the cytoplasm (hence their usual name.91) Erythrocytes containing granules (nuclear remants) that stain dark blue with vital stains such as brilliant cresyl blue and Evan’s blue.91 Reticulocytes Polymorphonuclear neutrophils (Fig. 9. granulocytes). Fig. Reticulocytes (Fig. Reticulocytes usually disappear within 4 hours after the release of the erythrocytes into the blood. is of diagnostic importance.

9. deep purple granules. pinkish. Lymphocytes and monocytes Lymphocytes and monocytes have a compact nucleus and may or may not have granules in the cytoplasm.94 Polymorphonuclear basophils . with scattered granules. 9. Cytoplasm: abundant. round. Polymorphonuclear eosinophils (Fig. 9. Fig. with densely packed dark purple chromatin. containing numerous large. Cytoplasm: very little visible. orange-red granules. Cytoplasm: very little visible. less densely packed than those of the eosinophils.9. Cytoplasm: very little visible. 9. Small lymphocytes (Fig. Sometimes the cell appears damaged.95) Size: 7–10 mm. The chromatin appears as a uniform deep purple mass. containing numerous very large. 9. Shape: round. mauve granules. Shape: round.92 Polymorphonuclear neutrophils Fig. Nucleus: large (occupying most of the cell). Nucleus: usually two lobes. 9.93) Size: 12–15 mm.94) These are the rarest type of granulocyte. Polymorphonuclear basophils (Fig. blue with no granules. round. densely packed. The granules appear brown-violet after staining. Small colourless vacuoles are sometimes present. Haematology 311 Fig. linked by strands of chromatin. containing numerous very small. Size: 11–13 mm.93 Polymorphonuclear eosinophils Nucleus: several (2–5) lobes. Nucleus: difficult to see because it is covered by the granules.

98 Plasma cells .95 Small lymphocytes Fig. 9. 9. 9. eccentric. usually reddish granules.96) Size: 10–15 mm. Rare or abnormal cells Plasma cells (Fig. tuberculosis.97) Size: 15–25 mm (largest of the leukocytes). Fig. with densely packed chromatin. pale blue. Shape: irregular. often in a wheel-like arrangement. Shape: round or oval. They may be seen in blood films prepared from patients with measles. Monocytes (Fig. 9.312 Manual of basic techniques for a health laboratory Fig. which are not easily seen. dark red granules. These masses are malaria pigment. Numerous very small vacuoles are present. Vacuoles are usually present. In patients suffering from malaria the cytoplasm often contains brownish-black masses. Cytoplasm: dark blue with a pale-staining area round the nucleus. Size: 12–15 mm. may lie to one side of the cell. Nucleus: round. 9. Nucleus: oval or round.96 Large lymphocytes Large lymphocytes (Fig. Fig. 9. Nucleus: variable.98) Plasma cells produce antibodies. often kidney-shaped with pale mauve chromatin arranged in strands. Cytoplasm: abundant. dust-like. 9. containing fine. other viral or bacterial infections or multiple myeloma. Shape: round or irregular.97 Monocytes Cytoplasm: pale blue. containing several large.

Hypersegmented polymorphonuclear neutrophils (Fig. except that their nuclei have 5–10 lobes and are often larger in size. with chromatin varying in colour from dark red to purple. Nucleus: round or irregular. especially infectious mononucleosis (glandular fever).99) are seen. 15–25 mm. They are also found in tuberculosis. forms a dark edge to the cell. There are also immature cells without granules and with nucleoli (lymphoblasts) (see Fig. Nucleus: large.100 Hypersegmented polymorphonuclear neutrophils Fig.100) Hypersegmented polymorphonuclear neutrophils look like normal neutrophils. If immature polymorphonuclear neutrophils (“band form” or “stab cells”) (Fig.101 Atypical lymphocyte . pale mauve. Atypical lymphocytes (Fig. containing 1–5 nucleoli. 9. Cytoplasm: usually darker blue than that of large lymphocytes.101) Atypical lymphocytes can be seen in viral infections. 9. severe malaria and the acquired immunodeficiency syndrome (AIDS). 9. Fig. 9. round. Size: very variable. in which the granules are very large and darkly stained. Cytoplasm: dark blue. with a clear unstained area round the nucleus. Shape: usually irregular.99 Immature polymorphonuclear neutrophils Fig. whooping cough and measles. Does not contain granules. 9. often lying to one side of the cell.102). nucleoli may be seen.9. Haematology 313 Immature granulocytes Immature polymorphonuclear granulocytes of the bone marrow pass into the bloodstream in severe bacterial infections. 9. caused by folic acid or vitamin B12 deficiency. 12–18 mm. Nucleus: without lobes. 9. They can be distinguished by the following features: Size: 12–18 mm. Size: large. Cytoplasm: pale blue or pink with many large mauve or dark red granules. Lymphoblasts can be seen in the blood films of patients with leukaemia. Such neutrophils can be seen in patients with macrocytic anaemia. report their number fraction as for other types of leukocyte. Toxic granulation may be seen.102) The earliest (most immature) of all the types of leukocyte. 9. Lymphoblasts (Fig. Does not contain granules.

and thrombocytes. 9. allowing sickling to take place. 2% solution (reagent no. 60–100 mm. If inherited from both parents it causes sickle-cell anaemia. Cytoplasm: contains numerous fine granules. which does not usually cause disease. such as Petri dishes Fresh sodium metabisulfite.3) found in the bone marrow. Nucleus: very irregular.1 Principle One drop of blood is mixed with one drop of sodium metabisulfite reagent on a slide. The cell wall is not clearly defined.2 Materials and reagents q q q q q q q q Microscope Microscope slides Coverslips Filter-paper Pasteur pipette (or dropping pipette) Two small wooden sticks Containers to prevent drying of the preparation. Size: very large.79). Haemoglobin S occurs mainly in tropical Africa but also in the Eastern Mediterranean region and among Americans of African origin.11 Test for sickle-cell anaemia Haemoglobin S is an inherited abnormal haemoglobin. The sickle-cell slide test does not distinguish between sickle-cell anaemia and sickle-cell trait. a serious disease. If the erythrocytes contain haemoglobin S. (Very rarely found in the blood. . 9.314 Manual of basic techniques for a health laboratory Fig.11. 9. mostly dark red.102 Lymphoblast Fig. 9.) 9. 9. If inherited from only one parent it causes sickle-cell trait.103 Megakaryocyte Megakaryocytes (Fig.11.103) The parent cells of thrombocytes (see section 9.1. 9. greatly lobulated but dense. they will become sickle-shaped or half-moon-shaped (see Fig. The reagent removes oxygen from the cells. 55).

It is important to examine several parts of the preparation. Positive result The erythrocytes become sickle-shaped or banana-shaped (Fig. Fig.106 Test for sickle-cell anaemia: negative result Fig.11. as sickling can occur more quickly in one part than in another. 9.107 Test for sickle-cell anaemia: positive result a: Sickle-shaped erythrocytes.3 Method 1.105). 9. If the test is negative. 9. 9.106). often with spikes (Fig. 3. Cover with a coverslip. . Add an equal-sized drop of sodium metabisulfite solution. 9. 9. re-examine the slide after a further 30 minutes. 9. Haematology 315 Fig. Place a small drop of capillary blood (about 4 mm diameter) in the centre of a slide (see Fig.104 Mixing the blood and sodium metabisulfite solution 9.65). Wait 30 minutes before examining the slide. 9. Negative result The erythrocytes remain round (Fig. 2.104). Note: When using a reducing reagent such as sodium metabisulfite it is not necessary to seal the preparation. Fig. 4. making sure that no air bubbles form.107 (b)).9. then after 2 hours and after 24 hours.4 Microscopic examination Examine the slide under the microscope using the ¥ 40 objective.107 (a)).105 Incubating the slide in a Petri dish 9. b: sickleshaped erythrocytes with spikes. Do not mistake normal erythrocytes lying on their side or crenated cells for sickle cells. 9. Support the slide on two sticks (Fig. 9. Mix carefully with the corner of a slide (Fig.11. Place the slide in a Petri dish that has wet filter-paper in the bottom.

They do not contain a nucleus. The number of reticulocytes in the blood indicates the degree of activity of the bone marrow in the production of erythrocytes. A blood film is stained with this dye and a certain number of erythrocytes observed under the microscope. either: — the number of reticulocytes per litre of blood. or — the proportion of erythrocytes that are reticulocytes is calculated.12. 13). target cells. If the test is positive a thin blood film should be examined. Patients with sickle-cell trait are not usually anaemic and have a normal erythrocyte morphology. nucleated erythrocytes. and when the marrow is very active (as in anaemia) their number increases. Other methods q The test can be carried out on venous blood provided it is freshly collected (within 1–2 hours of the test) or collected into an anticoagulant (EDTA dipotassium salt. From this observation. — concentrations of haemoglobin S are low. marked poikilocytosis and often macrocytosis.1 Principle The fine granules in reticulocytes can be stained with brilliant cresyl blue. 9.12.316 Manual of basic techniques for a health laboratory Note: False-negative results may occur if: — outdated reagents are used. if available Saturated solution of brilliant cresyl blue (reagent no. Whenever possible. The test can also be carried out using a test-tube rather than a Petri dish. deep-violet granules arranged in a network (reticulum). electrophoresis of the haemoglobin should be carried out to confirm a diagnosis of sickle-cell disease. 10% solution (reagent no. . 9. 22)). q 9. Commercial reagents are available for this method. — patients have moderate or severe anaemia. Patients with sickle-cell anaemia have sickle cells.2 Materials and reagents q q q q q q q q q q Microscope Microscope slides (grease-free) Glass spreader Test-tubes Test-tube rack Funnel Filter-paper Two Pasteur pipettes with teats Hand tally counter. This can be done in a reference laboratory. Reticulocytes contain fine.12 Determination of the reticulocyte number concentration/fraction Reticulocytes are immature erythrocytes that pass into the bloodstream from the bone marrow.

Filter a little of the cresyl blue solution into a test-tube. 9. This can be done by placing in the eyepiece a small circular piece of stiff black paper in which a hole of about 5 mm in diameter has been punched. 3. In the bottom of another tube place two drops of filtered cresyl blue solution (Fig. Depending on the practice in your laboratory or the specification of the requesting physician.0%. 9.1 1 Traditionally. Leave for 15 minutes. 6.109). 9.9.4 Microscopic examination Examine the smear using the ¥ 100 oil-immersion objective (Fig.2–2.110). If 500 erythrocytes are observed on the blood film and n of them are reticulocytes. the percentage of erythrocytes is calculated by multiplying n by 0.3).3 Method 1. . where the erythrocytes should be well separated from each other. 2.109 Collecting a capillary blood sample 9. Look at the end of the smear. 5. Example: Of 500 erythrocytes examined.0%.10.2 = 5%. Make a thin smear of the mixture with the spreader (see section 9. Haematology 317 Fig. or use venous blood collected in EDTA dipotassium salt solution and mix well.) Some haematologists prefer reticulocytes to be reported in terms of the number concentration (number of reticulocytes per litre of blood). Examine at least 100 erythrocytes.2. 25 are reticulocytes. Take the tube and shake it gently. 9. Erythrocytes stain pale blue. Keep a careful count of the total number of erythrocytes examined and the number of these that are reticulocytes. Leave the smear to air-dry. Place it on a slide ready for spreading. of the erythrocytes that are reticulocytes). Mix by gently shaking the tube. Remove one drop of the mixture. Collect a few drops of blood from the patient’s finger with a Pasteur pipette (Fig. The normal range for newborn infants is 2. while others prefer them to be reported in terms of the number fraction (the proportion of erythrocytes that are reticulocytes).e. 9.0–6.12. the proportion. make the appropriate calculation. reticulocytes have been reported in the form of percentages (i.12. The percentage of reticulocytes is then 25 ¥ 0. Add two drops of blood to the tube containing cresyl blue solution. 4. and that for adults and children is 0. Plug the tube with non-absorbent cotton wool. 9.108). (Counting is easier if the size of the microscope field is reduced.108 Preparing the cresyl blue solution Fig. expressed as a percentage.

they occur in most of the erythrocytes. Other structures that can be seen in blood films stained with brilliant cresyl blue The blood film stained with brilliant cresyl blue that is used for determining the reticulocyte number concentration and reticulocyte number fraction may also show the following structures. Reference range Table 9. deep violet granules. 9. the calculation will have to be adjusted accordingly. by age group Age group Infants (newborn) Children Adults a Reticulocyte number concentrationa 100–300 ¥ 109/l 8–110 ¥ 109/l 8–110 ¥ 10 /l 9 Reticulocyte number fraction 20–60 ¥ 10-3 2–20 ¥ 10-3 2–20 ¥ 10-3 Approximate values. c: mature reticulocytes (containing only a few granules). If n is the number of reticulocytes seen in examining 500 erythrocytes. They are found in a-thalassaemia or haemoglobin H disease.318 Manual of basic techniques for a health laboratory Calculation To calculate the reticulocyte number concentration. you must know the total erythrocyte number concentration. If C is the total erythrocyte number concentration (omitting the “¥ 1012/l”) and n is the number of reticulocytes seen on observing 500 erythrocytes.5 ¥ (2 ¥ 6) ¥ 109/l = 4.5 ¥ 1012/l number of reticulocytes seen in counting 500 erythrocytes = 6 reticulocyte number concentration = 4. b a c Fig.5 ¥ 12 ¥ 109/l = 54 ¥ 109/l (report this result). containing fine. Unlike the reticulum of the reticulocytes. Table 9. the reticulocyte number concentration is C ¥ 2n ¥ 109/l. Haemoglobin H bodies Haemoglobin H bodies appear as pale blue dots. To calculate the reticulocyte number fraction you do not need to know the erythrocyte number concentration.110 Examining the smear under the microscope a: Typical reticulocytes. Example: number of reticulocytes seen in counting 500 erythrocytes = 6 reticulocyte number fraction = (2 ¥ 6) ¥ 10-3 = 12 ¥ 10-3 Note: If more than 500 erythrocytes are examined on the blood film.7). variable in size. Example: total erythrocyte number concentration = 4. b: reticulocytes containing filaments. The concentration depends on the erythrocyte number concentration (see Table 9. . the reticulocyte number fraction is 2n ¥ 10-3.11 Reticulocyte number concentrations and reticulocyte number fractions.11 shows the reference ranges for different age groups.

They occur in glucose-6-phosphatase dehydrogenase deficiency following treatment with certain drugs. and the proportion of each type is reported as a percentage (e. 9. proceed as follows: Draw up a table with: — five vertical columns (N. check that the leukocytes are evenly distributed. E. go on to the next. Reference range Table 9. 9.13. the leukocyte type number fractions are called the “differential leukocyte (or white cell) count”.12.59. etc. 28 becomes 0. These totals give the percentage of each type of leukocyte. Thus. you know that you have counted 100 leukocytes..13 Determination of the leukocyte type number fraction 9. eosinophils 12%. variable in size. . Then add up the total for each vertical column.3) Paper Pencil. express the result in terms of the number concentration (i. The proportion of each type of leukocyte is reported as a decimal fraction.06 and basophils 0. L and M).25. 1 becomes 0. 9. eosinophils 0.111).01. The total of all the fractions should be 1. Thus 59 becomes 0.1 Example: neutrophils 0. monocytes 0.2 Materials q q q q q Microscope Immersion oil Well-spread thin blood films stained with a Romanowsky stain (see section 9.01. When 10 strokes have been made in the first line.13. lying to one side of the erythrocyte near the cell membrane.56. monocytes 6% and basophils 1% in the example given above). 8 becomes 0. lymphocytes 25%.12 shows the reference ranges for different age groups.28. 1 In the traditional system. To record the different types of leukocytes as they are counted.e.g.3 Microscopic examination Using the ¥ 100 oil-immersion objective.08. neutrophils 56%. and — 10 horizontal lines (see Fig. B. If the total leukocyte count is known.1 Principle A total of 100 leukocytes are counted and the number of each type seen is recorded. The totals are turned into decimal fractions by placing a decimal point two digits to the left (in some cases this may necessitate the insertion of a zero). lymphocytes 0. when the 10th line has been completed. number of cells per litre) rather than as a decimal fraction. the neutrophils may have collected at the end of the film.10. In a badly spread film.9. 9. Haematology 319 Heinz bodies Heinz bodies appear as blue granules. These decimals are the number fractions of each type of leukocyte and are the results that are reported when SI units are used.13. as in the last line of the illustration.

Abnormal findings q Fig.g.01 0.55 0. It can also be caused by allergies.02–0. ascariasis). M: monocytes.35 0. q Table 9.111 Table for recording the different types of leukocytes N: neutrophils.00–0. by age group Age group Neutrophils Newborn infants Infants (up to 1 year. malaria. typhoid fever. q Lymphocytosis is an increased proportion of lymphocytes (above 0.65). certain chronic infections (e.g.42 neutrophil number concentration = 0. E: eosinophils.05 0.30–0.03–0. children over 10 years and adults).g.48 Monocytes 0. Eosinophilia is an increased proportion of eosinophils (above 0.02–0.00–0. hookworm. Monocytosis is an increased proportion of monocytes (above 0.10 0. number of cells per litre) instead of as a number fraction.00–0.02–0. The other pattern shows a majority of neutrophils (seen in newborn infants. tuberculosis) and some toxic conditions.02–0.12 Normal leukocyte type number fractions.40–0.1 ¥ 108/l. as percentages). 9.06 To obtain values in traditional units (i.45 0.05).48 0.38–0. The differential leukocyte count is calculated by multiplying the percentage of a particular type of leukocyte (e.02–0.54 0.04 0. Neutrophilia is an increased proportion of neutrophils (above 0.65 0. malaria.01 0. infectious mononucleosis) and certain parasitic infections (e.06 0.05–0.g.05 0.55–0.06).00–0. sepsis) and some other diseases.00–0.42 ¥ 5 ¥ 109 = 2.06 0.05 Basophils 0. Lymphopenia is a decreased number of lymphocytes and may occur in AIDS.48 0.01 Lymphocytes 0.03–0.320 Manual of basic techniques for a health laboratory There are two main patterns of distribution of different types of leukocyte: q One pattern shows a majority of lymphocytes (this type is seen in infants and children under 10 years). multiply each value by 100.40–0. schistosomiasis. kala-azar (visceral leishmaniasis)). q q q .03–0. filariasis. It is particularly common in infections. It may occur in certain infections (e.01 0.45 in children). neutrophils) by the total leukocyte count and dividing by 100.06 0. The number concentration is calculated by multiplying the number fraction of a particular type of leukocyte by the total leukocyte number concentration. Neutropenia is a decreased number of neutrophils.35 in adults and above 0. B: basophils.g.g. q Each type of leukocyte may be reported in terms of its number concentration (i.e. It is found in certain virus infections (e.03–0. It almost always suggests a parasitic infection localized in the tissues (e.45–0.36–0.01 0. Example: total leukocyte count = 5000/mm3 percentage of neutrophils = 42% “absolute” neutrophil count = (42 ¥ 5000)/100 = 2100/mm3.25–0.55–0. Example: leukocyte number concentration = 5 ¥ 109/l neutrophil number fraction = 0.44–0.g.35 0.04 0. L: lymphocytes. excluding newborns) Infants (1–4 years) Children (10 years) Adults a Cell typea Eosinophils 0.65 0.e. It occurs in certain bacterial infections (e. measles).

Haematology 321 9.1 ¥ 105 1. If the erythrocyte number concentration (see section 9. If not.0 ¥ 105 .6 ¥ 105 2. can be based on a ratio of approximately one thrombocyte per 500–1000 erythrocytes being normal.10. Calculate the ratio of thrombocytes to erythrocytes.14 Determination of the thrombocyte number concentration 9. Reference range Table 9.13 shows the reference ranges for different age groups.5–5. Table 9.14.1 Materials q q q Microscope Immersion oil Well-spread blood film stained with a Romanowsky stain (see section 9.2 Microscopic examination Using the ¥ 100 oil-immersion objective.3). count the number of thrombocytes in 20 fields and make a rough estimate of the number of erythrocytes per field. 9.13 Normal thrombocyte counts. “high” or “low”.5–6. by age group Age group Infants (< 1 year) Children (1–15 years) Adults Thrombocyte count (per mm3) 3.7–4.14. either “normal”. a rough estimate of the thrombocyte count. the thrombocyte count can be estimated.5) is known.9.

5.0. it can be assumed that the patient’s results will also be correct.1. Use it with care. If venous blood is used. 5. 0. 10.1 Principle The proteins are first precipitated by trichloroacetic acid. plasma or serum.0 ml Water-bath at 100 °C Glucose reagents (reagent no.1. 10.8 ml of trichloroacetic acid solution into each of three centrifuge tubes. Blood chemistry 10.4). it is advisable to use fluoride oxalate (reagent no. 0.3 Method 1.1. 26) as the anticoagulant.1% solution — glucose stock reference solution (100 mmol/l) — glucose working reference solutions (2. Note: Trichloroacetic acid is corrosive. If the result of the test with the control serum is correct. q q 10. This will prevent the glucose from being destroyed in the blood.10. In patients with diabetes glucose is usually found in the urine (see section 7. 30) — trichloroacetic acid.5. 10. taken from a fasting patient2 Control serum.5 ml. 0.4). A control serum should be used with each batch of tests.2 Materials and reagents q q q q q q q Colorimeter Conical centrifuge tubes and large test-tubes (to hold 20 ml) Test-tube racks Blood (Sahli) pipettes.3. 1 2 This method is also used for estimating the glucose concentration in CSF (see section 8. Pipette 1. 322 . The glucose in the filtrate reacts with the o-toluidine reagent to give a green colour. 3% solution — o-toluidine reagent — benzoic acid. 20 and 25 mmol/l) Whole blood (capillary or venous).1 Estimation of glucose concentration in blood: o-toluidine method1 Estimates of the glucose (sugar) concentration in blood are required to help in the diagnosis of diabetes mellitus or any other condition in which there is abnormal carbohydrate metabolism in the body.2 ml Pipettes. which is measured using a photoelectric colorimeter.2.

Centrifuge the three tubes at 3000g for 5 minutes.10. deliver 0. The trichloroacetic acid solution will become cloudy where it makes contact with the blood specimen. 6. q Reference: — 0. 9. 10. With a 0.1 Delivering blood under trichloroacetic acid solution using a blood pipette A: trichloroacetic acid solution. 10. 8.8 ml). q Patient: — 0.2 ml of distilled water and 0.3).5 ml of o-toluidine reagent.1). When this test is performed using CSF.2 Rinsing the blood pipette with trichloroacetic acid solution Fig.2 ml blood pipette. 10. 10. These tubes will be used to prepare the reagent blank and the glucose working reference standard. as described in step 2. Pipette into each tube as follows: q Blank: — 0.5 ml of o-toluidine reagent.2). 10.4: — blank tube (B) — reference tube (R) — patient tube (P). Note: The o-toluidine reagent is corrosive.3 Expelling trichloroacetic acid solution into a centrifuge tube 2.5 ml of from the third centrifuge tube — 3. 10. Blood chemistry 323 Fig. P: patient tube.2-ml blood pipette.4 Labelling test-tubes for the test B: blank tube. Fig. 5. 10. Take three (or more if needed) large test-tubes and label as shown in Fig. The precipitated proteins in the tube containing the blood specimen will sediment and a clear supernatant fluid will be obtained. respectively. 1 Fig. .2 ml of glucose working reference solution to the second and third centrifuge tubes. R: reference tube.1) — i. 7. B: blood. 10.5 ml of o-toluidine reagent. 3. Raise the pipette and draw clear trichloroacetic acid solution into it in order to wash out all traces of the blood specimen (Fig. the volume required in this step is greater (0. deliver 0. respectively. Note: If more than one estimation is being carried out. Using a clean 0. 10.e.5 ml of supernatant fluid from the first centrifuge tube — 3.2 ml of the blood1 specimen to the bottom of the first centrifuge tube (B in Fig.5 ml of fluid from the second centrifuge tube — 3. 4. Expel the trichloroacetic acid solution from the pipette into the centrifuge tube (Fig. Mix well (the entire solution will become cloudy) and allow to stand for 5 minutes. under the trichloroacetic acid solution (A in Fig. label each of the P tubes with the name or number of the patient.

Place all the tubes in the water-bath at 100 °C for exactly 12 minutes (Fig. (a) Place the orange-red filter in the colorimeter. make the calculation for that serum in exactly the same way. ( ) 1 The calculation given is for SI units. (c) Adjust the reading of the colorimeter to zero with the cuvette containing solution B in place. prepare a calibration graph using the different concentrations of the glucose working reference solution treated as described in steps 6–9. place the cuvette in the colorimeter and read the absorbance. and fill the cuvette with solution R.4 Results Calculation Calculate the concentration of glucose in the blood specimen using the following formula:1 concentration of glucose in blood (mmol l) = AP AR ¥ C where: AP = absorbance reading of the patient’s specimen AR = absorbance reading of the glucose working reference solution C = concentration of the glucose working reference solution.5 Heating the testtubes in a waterbath 10. rinse the cuvette with a small amount of solution R (reference). High and low values If unusually high or low values are observed. (b) Fill the colorimeter tube or cuvette with the solution contained in the tube marked B (blank) and place in the colorimeter.324 Manual of basic techniques for a health laboratory 10. Reference range The reference ranges of glucose concentrations in the blood of fasting patients are shown in Table 10. 10. Prepare a new graph whenever the o-toluidine reagent is renewed. the test should be repeated in order to confirm the results. Mix the contents of each tube. 10. and fill the cuvette with solution P. place the cuvette in the colorimeter and read the absorbance. (d) Pour solution B out of the cuvette. The graph should be linear up to the highest concentration and should pass through the origin. Fig. AP. The formula for calculating blood glucose concentrations in traditional units is as follows: concentration of glucose (mg 100ml ) = concentration of glucose (mmol l ) ¥ 1 0. to confirm the linearity.1.1. AR. rinse the cuvette with a small amount of solution P (patient). (e) Pour solution R out of the cuvette. 12. as described below.5). Remove the tubes and allow them to cool in a beaker of cold water for 5 minutes. Measure the colour produced in a colorimeter at a wavelength of 630 nm. Note: If a control serum has been included. Calibration of the colorimeter Before taking measurements.0555 . pour this out. 11. pour this out. substituting Ac (absorbance of the control solution) for Ap in the formula.

Glucose concentrations lower than 2. is filtered out by the kidneys and excreted in the urine.3–5. 10. and in step 2 use 0. Perform the test and calculate the result exactly as before. 10.1 ml). Blood chemistry 325 Table 10. 50 ml. oxidizing reagent and thiosemicarbazide to give a red solution. It passes into the blood. If the kidneys do not remove urea. Recalculate the glucose concentration. set the colorimeter reading to zero. using the new value of AP and the value of AR that was obtained previously. serum or plasma (instead of 0. Divide the result by four to obtain the true glucose concentration.1 ml. The urea in the filtrate reacts with diacetyl monoxime in the presence of acid. The colour is measured using a photoelectric colorimeter.4 3. 5 ml Measuring cylinder. In step 1.4 Glucose concentration Traditional units (mg/100 ml) 60–100 70–100 70–115 70–115 Glucose concentrations higher than 16. 62): — trichloroacetic acid. 6) q . 0. 0.8 ml). 10.5 3.10. This can happen if the kidney tubules become damaged or if the volume of blood flowing through the kidneys is reduced.5 ml. use 1.9–6.9–5.2 Estimation of urea concentration in blood: diacetyl monoxime/thiosemicarbazide method Urea is a waste product formed in the liver following the breakdown of protein.2.2 Materials and reagents q q q q q q Colorimeter Conical tubes and test-tubes (to hold 20 ml) Pipettes.5 3. Using diluted solution B in the cuvette.3 mmol/l Repeat the entire test. 50 ml Water-bath at 100 °C Urea reagents (reagent no.2.4 ml of blood. Then read the absorbance AP with diluted solution P in the cuvette.9–6. the concentration in the blood is increased.1 Blood glucose concentrations in fasting patients Fluid SI units (mmol/l) Venous blood Capillary blood Serum Plasma 3.1 Principle The proteins are first precipitated by trichloroacetic acid.5 mmol/l Dilute solutions B (blank) and P (patient) with an equal volume of glacial acetic acid. Multiply the result by two (because solution P has been diluted 1 in 2) to obtain the true glucose concentration. 5% solution — diacetyl monoxime stock solution — colour reagent — urea stock reference solution (125 mmol/l) — urea working reference solution (10 mmol/l) Acid reagent (reagent no.6 ml of trichloroacetic acid solution (instead of 1.

3 Method 1. 2. Pipette into each tube as follows: q Blank: — 0. 11) Patient’s blood (treated with EDTA dipotassium salt. q 10. 5. 10. Centrifuge at high speed (3000g) for 5 minutes to sediment the precipitated proteins and obtain a clear supernatant fluid. (c) Adjust the reading of the colorimeter to zero with the cuvette containing solution B in place. Prepare the colour reagent immediately before use. pour this out. Mix the colour reagent in a large test-tube or small flask. 22)). Pipette into a conical centrifuge tube 50 ml of whole blood (treated with EDTA dipotassium salt solution). place the cuvette in the colorimeter and read the absorbance. Add 1 ml of trichloroacetic acid solution and mix. AP.326 Manual of basic techniques for a health laboratory q q Blank reagent (reagent no. using a 1:1 mixture of the diacetyl monoxime stock solution and acid reagent.5) to allow the red colour to develop. and fill the cuvette with solution R. Note: If more than one estimation is being carried out.0 ml of freshly prepared colour reagent. (e) Pour solution R out of the cuvette. 7. q Reference: — 0.1 ml of working reference solution — 3. (b) Fill the colorimeter test-tube or cuvette with the solution contained in the tube marked B (blank) and place in the colorimeter. (a) Place the green filter in the colorimeter.2. rinse the cuvette with a small amount of working reference solution R (reference). pour this out.4: — blank tube (B) — reference tube (R) — patient tube (P).0 ml of freshly prepared colour reagent. 10% solution (reagent no. label each of the P tubes with the name or number of the patient.0 ml of freshly prepared colour reagent. 3. Remove the tubes and place them in a beaker of cold water until they have cooled to room temperature. serum or plasma. 4. place the cuvette in the colorimeter and read the absorbance. Measure the colour produced in a colorimeter at a wavelength of 520 nm. . and fill the cuvette with solution P.1 ml of supernatant fluid — 3. A control serum (of known concentration) should be used with each batch of tests. Take three (or more if needed) large test-tubes and label as shown in Fig. Mix the contents of each tube. AR. it can be assumed that the patient’s results will also be correct. Prepare at least 15 ml of colour reagent for each test. rinse the cuvette with a small amount of solution P (patient). Place all the tubes in the water-bath at 100 °C for 15 minutes (see Fig.1 ml of blank reagent — 3. q Patient: — 0. 8. 6. (d) Pour solution B out of the cuvette. serum or plasma Control serum. If the result of the test with the control serum is correct. 10.

The formula for calculating blood urea concentrations in traditional units is as follows: urea concentration (mg 100ml ) = urea concentration (mmol l ) ¥ 1 0. serum or plasma in step 2.4 Results Calculation Calculate the concentration of urea in the blood as follows:1 urea concentration (mmol l) = Ap AR ¥ C where: AP = absorbance reading of patient’s specimen AR = absorbance reading of urea working reference standard C = concentration of working reference standard (10 mmol/l).2. using 0. repeat the entire test. High values If a value greater than 25 mmol/l (150 mg/100 ml) is obtained. but divide the result by two to obtain the true urea concentration.10. Blood chemistry 327 10.1 ml of whole blood (treated with EDTA dipotassium salt solution). Perform the test and calculate the results exactly as before. ( ) 1 The calculation given is for SI units. Reference range The reference range of urea concentrations in blood is approximately 3–7 mmol/l (18–42 mg/100 ml).167 .

which are not caused by a microorganism but can be detected by some of the diagnostic techniques applied in immunology. In some cases microorganisms are difficult to culture and isolate or may require special and often expensive techniques that are not available for routine diagnosis. the choice should be based upon the question asked and the availability of facilities. The cell-mediated system is associated with T-lymphocytes which can process and destroy foreign bodies. In neonates. These barriers are there to prevent entry of pathogens into the body. A number of diagnostic techniques based on biological reactions using antigen– antibody interactions are described.1 Antibodies Antibodies are found in serum. these immunology tests do not detect the microorganism directly. Most infectious diseases are diagnosed by isolating and identifying the infectious microorganism in a specimen from the patient. the skin and mucous membranes. The diagnostic techniques described here are those most frequently used as an aid in the diagnosis of certain diseases that cause an immunological reaction. Note that various approaches are available for different diagnostic purposes. which will help in understanding some of the immunological techniques described. In those disease states where a microorganism is involved. but provide evidence of its presence.11. saliva. which are the precursors of plasma cells. The aim here is to introduce some terminology and general concepts of the immune system. It is beyond the scope of this manual to go into detail about the immune system. Plasma cells produce and secrete protein substances known as antibodies or immunoglobulins. but some pathogens do manage to enter the body where they are immediately destroyed by phagocytic cells such as macrophages. antibody production is virtually absent and protection is provided by maternal antibodies mainly through breast milk and those that cross the placenta.1 Introduction to immunology The role of the immune system is defence. urine and other body fluids. often classified as autoimmune diseases. In other immunological disorders there is no microorganism per se to identify or isolate. The non-specific defences are physical or mechanical barriers. When pathogenic microorganisms enter the body the specific defence mechanisms are activated.1. There are also some “naturally” occurring immune diseases. The immune system has both nonspecific and specific mechanisms to recognize and respond accordingly to foreign and potentially pathogenic microorganisms. Immunological and serological techniques Many of the diagnostic techniques applied in immunology are based on the fact that antigens and antibodies interact. The specific mechanisms are divided into the humoral (antibodymediated) and the cell-mediated systems. for example. These barriers usually work very well. Several of these techniques detect specific metabolic products or specific antibodies and antigens. 11. tears. A growing infant is constantly exposed to various environmental antigens which 328 . 11. milk. The humoral system is associated with cells known as B-lymphocytes.

where amino acid sequences are highly variable. Antigens have sites on them known as antigenic determinants which can be recognized by antibodies.11. An individual’s immune system is normally capable of distinguishing substances belonging to the body from those that do not (self from non-self ). Complex proteins make “better” antigens than repeating polymers of lipids. 11.2 Antigens Antigens are molecules (usually proteins) that can elicit an immune response. IgE and IgD. Complexity The more complex the molecule. Relative molecular mass The relative molecular mass or size of an antigen also affects its immunogenicity. These proteins differ in their electrophoretic mobility. but not if administered intravenously.1 Structure of immunoglobulins 11. Immunological and serological techniques 329 promote production of antibodies. these are briefly discussed below. where amino acid sequences are very similar. relative molecular mass.e. the better the immune response to the antigen. antigenic structure. Within these H and L chains there are regions known as constant regions. In humans there are five major classes of antibodies or immunoglobulins: IgG. IgM. Certain conditions incapacitate the self-tolerance mechanism and the system starts to react against itself. large molecules make good antigens (i. 11. As a general rule. Stability It is essential for the antigen to be stable for it to be recognized by the immune system.1. Small molecules are usually poor antigens. provided they are foreign to the body. Route of antigen administration and dosage Antigens must be administered correctly. Recognition of the antigen as a foreign substance The most important property of an antigen is that which allows the body to recognize the substance as foreign. H chain L chain Fig. All the immunoglobulins have a structure composed of four polypeptide chains with two long or heavy (H) chains and two short or light (L) chains (Fig. carbohydrates and nucleic acids. Degradability The substance must be degradable. Some substances elicit an immune response if administered subcutaneously. The . and variable regions (usually at the ends of the chains). In order to initiate an immune response the immune system must be able to process the substance. sedimentation coefficient. The variable regions give the different antibodies their specificity. Antigens may have several determinants of different configuration or several of the same configuration such that antibodies of the same kind or of several different kinds can bind to these sites. shape and other properties. Several properties can influence the immunogenicity of an antigen. they are effective in generating an immune response). IgA.1).

2 Principle of radioimmunoassays 1 The analytical sensitivity and specificity of an immunological test must not be confused with the diagnostic sensitivity and specificity as defined to discriminate between diseased and nondiseased populations. However. This is a very sensitive approach for which a label is needed to detect the binding reaction.3 Antigen–antibody interactions The binding between an antigen and an antibody can be compared with the exclusive fit that exists between a lock and its key. . secondary and tertiary binding reactions.330 Manual of basic techniques for a health laboratory right dose is also important since too much or too little antigen may not elicit the desired immune response. The analytical sensitivity of an immunological test refers to its ability to detect small quantities of antigen or antibody. The analytical specificity of an immunological test is its ability to measure only the substance it purports to measure. 11. either an antigen or an antibody is conjugated with a radioactive tracer substance and the radioactivity is measured with a scintillation counter (Fig.1 Primary binding tests This group of tests provides a direct measure of the initial binding reaction between an antigen and an antibody. level of expertise required.2). Incubate then wash Incubate then wash R R R R R R R R R R R R R R R R R Adsorbed antibody Antigen-containing serum Radiolabelled antibody Fig.1 These are important considerations. especially when choosing a new test. Only primary and secondary binding reactions are described here.2 Principle of immunochemical techniques Antigen–antibody reactions can be classified into three groups: primary. To avoid these unwanted interactions. cost.1. availability. Tertiary reactions follow secondary reactions and usually occur in vivo. The antigen determinant (the “lock”) has a predetermined conformation and only a specific antibody (the “key”) with the appropriate variable regions (grooves and contours) will give a perfect fit. Radioimmunoassay In a radioimmunoassay. 11. It is used as a synonym for the limit of detection.2. causing cross-reactivity. the situation is not always so clear-cut. speed and simplicity. 11. enzyme immunoassays and immunofluorescence assays. 11. This type of assay is becoming less common. partly because of the need for radioactive substances and also because the measuring equipment required is difficult to use. Others include: applicability in a given laboratory environment. 11. Sometimes an antigen will combine (poorly) with an antibody that was produced against a different antigen. it is important to know and define the analytical sensitivity and specificity of immunological tests based on antibody–antigen reactions. Such tests include radioimmunoassays.

This change can be detected visually or with a spectrophotometer. Immunological and serological techniques 331 Enzyme immunoassay In an enzyme immunoassay.4 Principle of non-competitive enzyme immunoassays .3 Principle of competitive enzyme immunoassays Incubate then wash Incubate then wash E + Enzyme substrate E E E E E Solid-phase adsorbed antigen Antibody-containing serum Enzyme-labelled anti-immunoglobulin E Colour Fig. The binding event can be either competitive or non-competitive.3). The enzyme immunoassays are replacing many radioimmunoassays because of their advantages over the latter. the conjugate would contain anti-immunoglobulin and when testing for an antigen. which include longer reagent shelf-life. The enzymes used in ELISAs include horseradish peroxidase. lysozyme and b-galactosidase. Incubate then wash Incubate then wash E E + Enzyme substrate E E E E Solid-phase adsorbed antigen Antibody-containing serum Enzyme-labelled anti-immunoglobulin Colour Fig. no restrictive regulations for reagent disposal. In non-competitive binding (sandwich technique). cheaper and simpler equipment. an antigen or antibody is conjugated with a labelled enzyme and a colour change is produced by the enzyme reacting with its substrate. The test sample containing the corresponding antibody or antigen is then added.4). and safer reagents. the conjugate would contain an antibody specific for that antigen. An example of this technique is the enzyme-linked immunosorbent assay (ELISA). The amount of binding of the conjugate is directly related to the amount of antigen or antibody in the test sample and only the bound portion is measured in this assay. 11. Competitive binding depends on competition between a labelled (known amount) and an unlabelled (unknown amount) antigen for the same antibody (when measuring antigen) or between a labelled (known amount) and an unlabelled (unknown amount) antibody for the same antigen (when measuring antibody) (Fig. alkaline phosphatase. 11. 11. 11. the antigen or antibody is adsorbed (or immobilized) to a solid phase. A labelled antibody or antigen (conjugate) is added last to form the top layer of the sandwich (Fig. which may be an insoluble particle (bead) or the sides of a test-tube or the bottom of a microtitre plate.11. When testing for an antibody. The amount of binding of the labelled antigen (or antibody) is related to the amount of unlabelled antigen (or antibody) present.

5 Principle of direct immunofluorescence F F Fluorescence F F F . In these tests one can actually see the effects of the binding event without the aid of an additional label. Incubate then wash F F F F F Antigen (cell or tissue specimen) Fluorescence-labelled antibodies Fig. Two types of immunofluorescence technique can be used.2. A fluorescent microscope. 11. which is a modified light microscope. precipitation. The indirect method is more sensitive than the direct method because it is amplified in the sense that each unlabelled antibody can be bound by two labelled antibodies.5). 11. 11. Indirect immunofluorescence Indirect immunofluorescence is used to determine whether antibodies are present in a patient’s serum. The excess energy is released in the form of light. complement-dependent reactions and neutralization methods. The agglutination and precipitation methods are routinely employed for diagnostic purposes. Direct immunofluorescence Direct immunofluorescence is used when testing for an antigen. is used to visualize the light emitted.2 Secondary binding tests The secondary binding tests enable visible manifestations following the primary reaction to be observed. The antigen and antibody react.6). Fluorescence occurs when molecules that have been excited to a higher energy state return to their normal energy state. The tissue specimen is examined under the microscope and fluorescence is seen where the antibody is attached to the antigen (Fig. These tests include agglutination. The serum is applied directly to an appropriate tissue specimen containing an antigen directed against the specific antibody under investigation. The labelled anti-immunoglobulin will attach to any antibody already bound to the antigen in the tissue specimen and shows up under the microscope as areas of fluorescence (Fig. An anti-immunoglobulin with a fluorescent label is added and the tissue specimen is then incubated before being washed again. then the tissue specimen is washed. The antiserum is applied directly to the tissue specimen. then the tissue specimen is washed. The antigen and antibody react. These methods are briefly described below.332 Manual of basic techniques for a health laboratory Immunofluorescence Fluorescent dyes such as fluorescein isothiocyanate and tetra-methylrhodamine isothiocyanate can be coupled to antibodies without destroying their specificity. In this technique a fluorescent dye is linked to the isolated portion of an antiserum containing antibodies directed against a specific component of cells or tissue. 11.

11.6 Principle of indirect immunofluorescence Incubate then wash Antigen (insoluble) Antibody-containing serum Fig. Active agglutination (direct) Active agglutination involves antigenic determinants that are an intrinsic constituent of the particle. bacteria. The presence of antibody in the serum causes agglutination of the particles (Fig.11. 11. The interaction of surface antigens and antibodies directed against them leads to cross-linking of adjacent particles. The erythrocytes are usually treated with tannic acid. which alters their surface properties so that the antigen can bind firmly. bentonite (clay) and polyvinyl latex where the antigen is simply adsorbed. to form a lattice of agglutinated cells. e. Immunological and serological techniques 333 Incubate then wash F F F F F F Antigen (cell or tissue specimen) Antibody Fluorescence-labelled anti-immunoglobulin Incubate then wash F Fig. Other insoluble particles include bacteria. Semi-quantitative titrations can be carried out to determine the amount of antibody present in a sample. A soluble antigen is combined with insoluble particles such as erythrocytes. 11.g.8) and the reaction is usually scored on a scale of 0 to 4 +.7).g. charcoal. 11. The antibody content is expressed as a titre — F Fluorescence F Precipitation F . haemagglutination reactions used for blood grouping. Passive agglutination (indirect) Passive agglutination involves antigenic determinants that are not an intrinsic constituent of the particle. e. A constant volume of suspended particles (antigen) is added to a constant volume of serially diluted antiserum.7 Principle of agglutination Agglutination Agglutination involves the reaction of an antibody with a particulate (insoluble) antigen leading to visible clumping of these particles (Fig.

in precipitation reactions the interaction is between a soluble antibody and a soluble antigen. antibody and antigen complexes cross-link and form a precipitate.9 Principle of the agglutination inhibition test for hCG the reciprocal of the final dilution of antiserum capable of producing visible agglutination.334 Manual of basic techniques for a health laboratory Incubate then wash Incubate then wash Antigen carrier particles Antigen (soluble) Absorbed antigen Antibody-containing serum Agglutination Fig. the absence of agglutination with a specimen under investigation indicates a positive test result.9). the antibody reacts with it and is not free to react further after the subsequent addition of indicator particles or cells. 11. . Thus. An example of this kind of assay is the detection of human chorionic gonadotropin (hCG) used in the test for confirmation of pregnancy and also in other pathological conditions where hCG levels are important (Fig. If soluble antigen is present in the sample. Precipitation methods can be quantitative or qualitative and the interactions are dependent on ionic strength.8 Principle of passive agglutination hCG antibody Adsorbed hCG antigen hCG antigen (urine specimen from pregnant woman) No precipitation = positive result (a) hCG antibody Adsorbed hCG antigen Precipitation = negative result (b) Fig. This assay is based on competition between particulate and soluble antigen for antibody-binding sites. Precipitation Unlike agglutination reactions in which the antigen is particulate (insoluble). The antibody and the test sample are allowed to react together. 11. If a soluble antibody is incubated with a soluble antigen. 11. Agglutination inhibition Agglutination inhibition is used for determining the presence of antigen. pH and concentration.

The assays are rapid and sensitive. radial immunodiffusion (Mancini technique).11. The concentration of unknown samples is calculated with the aid of a standard curve which is prepared by plotting the diameter2 of the resulting ring of precipitate produced by a series of antigen solutions of known concentration (Fig. Diameter2 of ring of precipitate Standard 1 Standard 2 Standard 3 Unknown Antigen (or antibody) concentration Fig. Nephelometry and turbidimetry Nephelometry and turbidimetry involve the measurement of the light scattering and light absorption properties. turbidimetry.11 Principle of radial immunodiffusion . In some assays. This technique can be used for the quantitative measurement of complement factors and immunoglobulins. These include nephelometry. The excess antibody zone in which all available antigenic determinants are bound by individual antibody molecules. excessive amount of antibody is incubated with an antigen in a cuvette. Immunological and serological techniques 335 Mass of precipitated antibody A quantitative precipitin curve can be drawn in which the proportion of antigen and antibody determines the extent of cross-linking and precipitation. double diffusion (Ouchterlony) and some immunoelectrophoresis techniques. 11.11). In nephelometry light is passed through the cuvette and the scatter produced by the antigen–antibody complexes that have been formed is measured. 11. leaving some molecules of antigen unbound. 11. These techniques are used to measure concentrations of proteins and drugs in serum or CSF. The antigen concentration is determined from a standard curve made by measuring the light scatter produced by a series of antigen solutions of known concentrations. The curve shows the following features (Fig. respectively. A constant. leaving some antibody molecules unbound.10): q Excess antibody Equivalence Excess antigen The equivalence zone in which the proportions of antigen and antibody are equivalent. In turbidimetry light is passed through the cuvette and the absorption produced by the antigen–antibody complexes that have been formed is measured. 11. A conventional photometer can be used for this purpose.10 A quantitative precipitin curve Several other immunological techniques employ the precipitation reaction in one form or another. q q Concentration of antigen Fig. Radial immunodiffusion (Mancini technique) Radial immunodiffusion is based on the principle that a quantitative relationship exists between the amount of antigen placed in a well cut in an agarose gel containing antibody and the diameter of the resulting ring of precipitate. of antigen–antibody complexes. The antigen concentration is proportional to the square of the diameter of the ring of precipitate. The excess antigen zone in which all the antigen binding sites of an antibody are bound by individual antigen molecules. polymers are added to accelerate the formation of antigen–antibody complexes.

examine the plates and compare the reactions of the test sera with those of the control sera (Fig. but the “standard” ASOT is based on the fact that streptolysin O will lyse human or sheep erythrocytes unless neutralized by anti-streptolysin O antibodies present in the patient’s serum. 7.12).3. The anti-streptolysin O test (ASOT) is the most commonly used laboratory test for following a streptococcal infection and its sequelae (rheumatic fever and acute post-streptococcal glomerulonephritis). If any sera are positive. wooden orange sticks or rotator Test-tubes.3. 11. 11. 11. repeat steps 3–6. or place on a rotator. . 3. The highest dilution of serum that causes agglutination is the titre.85% solution (reagent no. 2. Shake the vial of latex RF reagent and add one drop to each of the samples on the test plates. 0. 5. Other approaches are now available.1 Materials and reagents q q q q q q Test plates (preferably with a dark background) Stirring rods.2 Method 1. 53). Apply one drop of each dilution to the test plates. b: negative result. Fig. Bring the test and control sera and latex RF reagent to room temperature. using a twofold dilution. 4.12 Latex agglutination test a: Positive result. 5 ml Test-tube rack Micropipettes Latex rheumatoid factor (RF) reagent (aqueous suspension of latex particles coated with human IgG) Negative and positive control sera Sodium chloride.4. 6. After 2 minutes. 11.4 Tests for the determination of anti-streptolysin O antibodies 11.336 Manual of basic techniques for a health laboratory 11. Dilute the test and control sera 1 : 5 with sodium chloride solution. Mix well with stirring rods or orange sticks (one for each sample) and rotate the plates gently (about 10 times). q q The above-mentioned materials and reagents are usually supplied as part of a commercial test kit.3 Determination of rheumatoid factors by the latex-agglutination technique 11.1 Anti-streptolysin O test (ASOT) Streptolysin O is a toxin produced by haemolytic streptococci.

For screening purposes.11. Control tube 14 should show complete haemolysis. Add 0. Mix and incubate in a water-bath at 37 °C for 45 minutes. 3.4.5 streptolysin O units. Mix and incubate in a water-bath at 37 °C for 15 minutes. q Method 1. the highest dilution of patient’s serum that still has a clear supernatant (no haemolysis) is the end-point and its Todd unit value (reciprocal of the dilution) is reported. From these master dilutions. 0. .1.e. After centrifugation at 500g. 5. the highest dilution) showing no haemolysis. 1000 ml Test-tubes. Principle The test is performed by incubating a constant amount of standardized streptolysin O with serial dilutions of heat-inactivated patient’s serum (containing antistreptolysin O antibodies) at 37 °C for 15 minutes. Control tube 13 should show no haemolysis. pH 6. 53) 5% Suspension of washed sheep erythrocytes in sodium chloride.5 ml of serum + 4. 0. 75 mm ¥ 12 mm Test-tube racks Serological pipettes Water-bath Centrifuge Phosphate-buffered water. Centrifuge the tubes gently at 500g for 3 minutes and observe for haemolysis. Add 0. If there is haemolysis in this tube the test should be repeated.2).85% solution (reagent no. The end-point is the last tube (i.5 ml (equivalent to 1 International Unit (IU)) of reduced streptolysin O to all the tubes except tube 13.5 ml of phosphate buffer = 1 : 10 0.5 ml of a freshly prepared 5% suspension of sheep erythrocytes when incubated for 1 hour at 37 °C. Materials and reagents q q q q q q q q q Volumetric flask.5 ml of 1 : 10 serum + 4. One Todd unit is defined as the amount of anti-streptolysin O antibody that will neutralize 0. Immunological and serological techniques 337 One streptolysin O unit is the minimum amount of streptolysin O that will lyse 0. Make three dilutions of the patient’s serum (inactivated by heating at 56 °C for 30 minutes) as follows: 0. 53) Reduced streptolysin O (instructions for the preparation of reduced streptolysin O from the unreduced form are usually provided by the manufacturer). This technique is rather time-consuming and simpler and more rapid methods using latex agglutination are now available (see section 11.8 (reagent no.85% solution (reagent no.5 ml of phosphate buffer = 1 : 100 1 ml of 1 : 100 serum + 4 ml of phosphate buffer = 1 : 500 2. 43) Sodium chloride. mixing again after the first 15 minutes of incubation. A freshly prepared suspension of 5% sheep erythrocytes is then added to all the tubes and incubation is continued for a further 45 minutes.5 ml of the 5% suspension of sheep erythrocytes to each tube. make an extended series of dilutions as shown in Table 11. 4. use only the first seven tubes and the control tubes (13 and 14).

4.g.5 0.5 0.6 0. examine the plates and compare the reactions of the test sera with those of the controls. add one drop to each of the test and control sera.6 0.5 1. Mix well with stirring rods or wooden sticks (one per sample) and rotate the plates gently about 10 times.5 0. Shake the anti-streptolysin O latex reagent to mix.1 Preparation of dilution series for the anti-streptolysin O test Tube number Volume of patient’s serum (inactivated) (ml). A positive reaction is indicated by the presence of agglutination.5 0. 6.5 0. The highest dilution that causes agglutination is the titre. repeat steps 2–5.5 0. wooden sticks or rotator Test-tubes.4 0.0 0. 50 ml Anti-streptolysin O latex reagent: suspension of latex particles coated with streptolysin O Negative control serum Positive control sera (strongly and weakly positive) Sodium chloride. 5.5 0. Apply one drop of each of the test and control sera to the test plates.4.6 0.5 0.85% solution (reagent no.5 0.2 0.8 0.8 0.5 0.0 0.2 — — 0.5 0.4 0.5 0.8 0.5 0.5 0. 3.5 0.4 0.5 0. q q q Method 1. diluted: 1 : 10 1 2 3 4 5 6 7 8 9 10 11 12 13 14 0.5 Volume of streptolysin O buffer (ml) Resulting serum dilution Volume of reduced streptolysin O buffer (ml) Volume of 5% suspension of sheep erythrocytes 11. Most anti-streptolysin O reagents have a detection limit (e.0 1 : 12. 0.2 — — — — — — — — — — — — 1 : 100 — — 1.3 — — — — — — — 1 : 500 — — — — — — — 1.2 Latex agglutination Materials and reagents q q q q q q Test plates Stirring rods.5 0. 53).0 0. 2.8 0.2 0. or place on a rotator. A negative reaction is indicated by the absence of agglutination.338 Manual of basic techniques for a health laboratory Table 11.5 1 : 50 1 : 100 1 : 125 1 : 167 1 : 250 1 : 333 1 : 500 1 : 625 1 : 833 1 : 1250 1 : 2500 control control 0.5 0.5 0.4 0. 200 IU/ml) that is usually multiplied by the dilution factor to give the serum concentration of anti-streptolysin O in IU/ml. using a twofold dilution.5 0.8 1.5 0.5 0.5 0.5 0.2 0.0 0.6 0.5 0. After 2 minutes. . Bring the reagents and test and control sera to room temperature. 5 ml Test-tube rack Micropipettes. If any sera are positive.0 0.5 0.7 0.

11. The above-mentioned reagents are usually supplied as part of a commercial test kit. A negative reaction (non-pregnant or b-hCG absent) is indicated by the presence of agglutination.5. Mix the latex hCG reagent suspension well. Mix carefully. 4. The results for this semi-quantitative analysis are reported in IU/ml which can be obtained by multiplying the dilution factor of the highest dilution that does not cause agglutination by the sensitivity or limit of detection of the method. as stated by the manufacturer. 2. 11. Bring the urine samples and reagents to room temperature. apply one drop to the test samples and rotate the plates or mix with stirring rods or wooden sticks (one per sample). 75 mm ¥ 12 mm Test-tube rack Anti-b-human chorionic gonadotropin (anti-b-hCG) antibody Latex hCG reagent (suspension of latex particles coated with hCG) Negative control Positive controls (strongly and weakly positive). 3. This may occur in both pregnant and nonpregnant patients. IgG and IgM by radial immunodiffusion 11.6 Quantitative determination of IgA.1 Materials and reagents q q q q q q q q Test plates Stirring rods. wooden sticks or rotator Test-tubes. Add one drop of anti-b-hCG antibody to each of the samples and each of the controls. Semi-quantitative analysis Semi-quantitative analysis may be desirable in some cases where the production of b-hCG may have a pathological cause. Immunological and serological techniques 339 11. A positive reaction (pregnant or b-hCG present) is indicated by the absence of agglutination.2 Method 1. Make a twofold dilution of the positive urine samples and examine as described in steps 2–5 above. Add one drop of each of the urine samples and each of the controls to the test plates.5 Determination of b-human chorionic gonadotropin (b-hCG) in urine by the agglutination inhibition b technique 11.1 Materials and reagents q q q Siliconized glass plates 8 cm ¥ 12 cm. After 3 minutes examine the plates and compare the reactions of the test samples with those of the controls.6.5. 5. The highest dilution that does not cause agglutination is the titre. or Petri dishes (glass or plastic) Boxes with tightly fitting lids Hole-punch with inner diameter of 2 mm .11.

0. q IgG: 1 : 20. 2. 1 : 16 and 1 : 40 dilutions of the patients’ sera in 0. Prepare two more agarose gels in the same way: the first with 0. 1 : 16. 1 : 64 and 1 : 128. 1 : 80. Calculate the exact volume of 1% agarose gel that needs to be prepared to cover all the plates with gel to a thickness of 1. 1 : 4. Punch holes of 2-mm diameter in the gels. allow it to cool to 56 °C.96 mg/ml (111 IU/ml). 1 : 32. for the determination of: q IgA: 1 : 8. IgA and IgG. 11. 1 : 8 and 1 : 16.340 Manual of basic techniques for a health laboratory q q q q q q q q q q q q Pipettes. Warm the antisera to 56 °C in the water-bath.6. 6. Prepare 1 : 2. 3. Add 0. 3% solution in distilled water Sodium chloride.1% sodium azide.15 mol/l sodium chloride for the determination of IgM. 3% solution. 1 : 40.2 ml of antihuman IgG antiserum per 10 ml of agarose solution and the second with 0. . q IgM: undiluted. 8.5 mg/ml (110 IU/ml) — IgM 0.2 Method 1. 1 : 2. Dissolve the agarose by placing it in the water-bath at 100 °C. 5.The formula for calculating this is as follows: pd 2 ¥ 0. Coat as many glass plates (or Petri dishes) as necessary (allowing one per patient) with agar. as follows. The above-mentioned reagents are usually supplied as part of a commercial test kit. When the suspension is clear.15 mol/l sodium chloride with 0. Mix thoroughly. 9.15 4 where d is the diameter of the plate (in cm). 4.13 ml of anti-human IgM antiserum per 10 ml of agarose solution. 10.5 mm and allow it to solidify at room temperature.15 mol/l sodium chloride. Pour on to each glass plate (or Petri dish) the exact volume of the agarose– antiserum mixture needed to make a gel with a thickness of 1. 7.5 mm.1 ml of anti-human IgA antiserum to each 10 ml of the agarose solution. 1 : 160 and 1 : 320. Prepare twofold dilutions of the standard serum in 0. 5 ml Water-bath Thermometer Test-tubes Test-tube racks Agar.15 mol/l with 0. respectively.1% sodium azide Agarose or agar Anti-human IgA antiserum Anti-human IgG antiserum Anti-human IgM antiserum Human standard serum containing: — IgA 2.0 mg/ml (123 IU/ml) — IgG 9. Prepare 40 ml of 1% agarose in 0.

11. If borderline results are obtained. Add the indicated amount of conjugate (enzyme-linked anti-human (usually goat) IgG) and incubate according to the manufacturer’s instructions. 1. 3. 12. reagents and controls.7. 11. repeat the test in case there have been technical errors. 7. The stopping solution inhibits any further reaction between the enzyme and the substrate.1 ELISA Materials and reagents q q q q q q q Micropipettes Incubator or water-bath at 37°C Washer or vacuum pump Spectrophotometer (reader) Distilled or deionized water ELISA test kit (commercially available) Solid-phase support system. Calculate the cut-off values for each test run according to the manufacturer’s instructions. the general steps are as outlined below. Method Each kit comes with its own instructions which should be followed carefully. Read the results in a spectrophotometer at the recommended wavelength. The washing should not remove the HIV antibodies that have attached to the solid phase during incubation. 14.7 Tests for the determination of HIV antibodies 11. 15. Make plots of the titrations of the standard serum. 13. At the end of the incubation period. Pipette 5 ml of each of the dilutions of the standard serum and the patients’ sera into different holes of the appropriate agarose gels (see steps 6 and 7). Place the plates in tightly closed boxes in a humid atmosphere and incubate them for 3 days at room temperature. Add the appropriate quantity of substrate and incubate according to the manufacturer’s instructions. repeat the test using a different ELISA and/or a rapid test system (see section 11. Add the test (serum) sample and controls to the antigen-precoated solid-phase support system and incubate at the specified temperature for the appropriate time. This is the colour development stage and should be protected from light.2). 11. examine the sample using a western blot. 2. Carefully aspirate the sample from the solid-phase system and wash to remove excess sample and other proteins. 6. On the x-axis plot the square of diameter of the rings of precipitate. However.11). 9. 5. Use these curves to read the concentration of IgA. Alternatively. and on the y-axis plot the concentrations of the standard serum (see Fig. 4. Measure the diameter of the rings of precipitate (in millimetres) using a ruler. add the stopping solution. 8. If borderline results are still obtained. Carefully aspirate the liquid again to remove any unbound conjugate and wash the solid-phase system.7. Immunological and serological techniques 341 11. . IgG and IgM in the patients’ sera.

especially in nurseries. A positive result is indicated by any colour development on the antibodycoated spot. Substrate Wash 11. reagents and positive and negative controls Weak-positive in-house control serum. which is commercially available.342 Manual of basic techniques for a health laboratory A test run is invalid if the positive control values are less than the calculated cutoff values. A spot should be visible on the positive control and no colour should be seen on the negative control. The test run is invalid if the results obtained using the controls are not as described above. contaminated needles and other contaminated materials. a polystyrene strip is coated with HIV antigen and allowed to react with serum. The antigen markers appear first or earlier on after exposure to the virus. q Incubate Method Follow the instructions provided in the kit. it is not routinely tested for. Fig.8 Tests for hepatitis virus infection Incubate Routine tests for hepatitis include the use of markers for hepatitis A. Hepatitis B and C viruses are transmitted through blood products. These tests are generally employed in situations where quick decisions may need to be taken and often require no equipment other than that provided in the kits. In the dipstick test for HIV antibody. 11. B and C viruses.13). Any HIV antibody present will then bind to the HIV antigen. however. 11. Weak-positive in-house controls should be included to help in reading results.7. except in cases of epidemics. 11. Hepatitis A is most common in children. After subsequent incubation with a substrate solution. Note: Criteria for testing samples using a western blot vary from laboratory to laboratory. In such cases. Materials and reagents Wash Dipstick c Test sample Incubate q q q Enzyme-conjugated antiserum Timer Absorbent towels or filter-paper Commercially available test kit containing dipsticks. In some cases a specific request may be made for a western blot even if the ELISA was non-reactive.13 Dipstick assay for HIV antibody . dry and evaluate visually — surface antigen (HBsAg) — antibody to surface antigen (anti-HBs) — envelope antigen (HBeAg) — antibody to envelope antigen (anti-HBe) Spot = positive reaction p No spot = negative reaction — antibody to core antigen (anti-HBc). Some laboratories test all samples that give a positive reaction in the ELISA. the test run must be repeated. a coloured spot that indicates the presence of HIV antibody will develop (Fig. body fluids. Hepatitis B virus has several markers which include: Wash. The concentrations of these markers vary during the course of an infection.2 Dipstick test Principle Dipstick tests were developed for the rapid detection of antigens and antibodies in human serum.

Add the test (serum) samples and controls to the anti-HBs precoated solidphase support system and incubate according to the manufacturer’s instructions. Hepatitis testing is routinely done by solid-phase ELISA and radioimmunoassay methods. If colour develops prior to application. In such cases the assay must be repeated. Make appropriate dilutions of reagents or specimens if required. Avoid cross-contamination of samples. Calculate the cut-off value for the test run as instructed by the manufacturer. temperatures and other conditions specified in the manufacturer’s instructions. Aspirate the liquid and wash to remove any unbound conjugate. Using a vacuum pump or automated washer. Make sure that the pre-coated antigen or antibody (solid phase) is not disturbed during the addition of the sample or of beads. Add the specified amount of conjugate (enzyme-linked anti-HBs) and incubate according to the manufacturer’s instructions. Precautions The ELISA method is fairly easy to perform. Add the specified amount of substrate (usually o-phenylenediamine) and incubate in the dark. Adhere strictly to the incubation times. q q q . The general principles of the ELISA technique for one of the markers for hepatitis B virus are outlined below. The stopping solution (usually an acid) inhibits any further reaction between the enzyme and the substrate.1 ELISA for hepatitis B surface antigen Materials and reagents q q q q q Micropipettes Incubator or water-bath Washer or vacuum pump Spectrophotometer (reader) Commercially available test kit containing solid-phase support system. reagents and controls Distilled or deionized water. 4. Store the solution in a closed container. The test run is invalid if the positive control values are less than the cut-off value. in the dark. 2. but pay attention to the following: q q q Make sure that the reagents and samples are brought to room temperature. carefully aspirate the liquid from the solid phase and wash the system.11. a new solution should be prepared. 8. 7. 11.) 6. Add the stopping solution as specified. (This is the colour development stage and should be protected from light.8. Prepare only enough chromogen solution for a single test run. Immunological and serological techniques 343 Seroconversion (antibody production) often occurs several weeks or months after exposure. 5. q Method 1. 3. Read the results in a spectrophotometer at the specified wavelength. Commercial kits for detection of hepatitis markers are available and specific criteria and instructions are provided with each kit.

15 Applying a test sample to the dipstick 4. Polyclonal antibodies against HBsAg are chemically fixed to another area of the strip (zone B in Fig. 11. Materials and reagents q Na me Zone B Zone A Fig.14). A drop of human serum is applied to zone A (Fig.16 Dipstick test for HBsAg: positive reaction q q q q The dipstick is pretreated with a mouse monoclonal antibody against HRP-II which is applied in a line across the stick about 1 cm from its base. 11.9 Dipstick test for falciparum malaria Na me Zone B Dipstick tests are also available for the detection of the malarial parasite Plasmodium falciparum. and form a visible red band in zone B (Fig. 11. 11. 2. Conjugates of monoclonal antibodies against HBsAg coupled to colloidal gold particles are adsorbed to one area of a nitrocellulose strip (zone A in Fig. Fig. The HBsAg antigen in the serum binds to the antibody conjugate and the gold–HBsAg immunocomplex migrates along the strip until it reaches the fixed polyclonal antibodies in zone B. 11. The polyclonal antibodies precipitate the gold–HBsAg immunocomplex. Label the test strip with the patient’s name and/or number. No red band is formed if the serum does not contain HBsAg. 3. .16).8.2 Dipstick test for hepatitis B surface antigen Principle The dipstick test for the detection of hepatitis B surface antigen (HBsAg) takes advantage of the formation of a visible spot by precipitating immunocomplexes.15).14). test cards. A second dotted line of HRP-II antigen is incorporated into the dipstick about 2–3 mm above the line of monoclonal antibody as a positive reagent control. Zone B Method Zone A 1. 11.9.14 Dipstick for the detection of HBsAg Nam e Commercially available test kit containing dipsticks.344 Manual of basic techniques for a health laboratory q In addition to the controls in the kit. it is generally recommended to include an in-house control of known optical density for quality control purposes. 11. Inspect zone B after 10–20 minutes for the appearance of a spot indicating a positive reaction. Zone A 11.1 Materials and reagents q Capillary tubes and rubber bulbs Test-tubes Test-tube rack Reaction stand Commercially available test kit containing dipsticks. Fig. 11. The test described here is based on the detection by monoclonal antibodies of the species-specific histidine-rich protein II (HRP-II) which is expressed by the asexual blood stages and possibly early gametocyte stages of the parasite. Add a drop of serum to zone A as recommended by the manufacturer. reagents and controls. reagents and controls. Allow the serum fluid to migrate to zone B on the test strip. 11.

9. 5.20).20 Applying detection reagent to the dipstick . Place the dipstick in the lysed blood until all the blood has been absorbed (Fig. This reagent consists of a suspension of micelles (phospholipid vesicles) containing sulfo-rhodamine B as a marker coupled to rabbit antibody raised against HRP-II.2 Method 1. 11. Apply one drop of detection reagent to the base of the dipstick (Fig. apply two drops of washing reagent to clear the lysed blood (Fig. 11. 6. 11.19 Placing the dipstick in the lysed blood Fig.21).19). 11. Collect a finger-prick sample of blood from the patient. Place one drop of the lysed blood sample into one of the wells of the test card in the reaction stand (Fig. If the result is positive a thin red line will be left across the dipstick with a broken line (the reagent control) above it. 11.18). 2. Place one drop of blood into a test-tube containing three drops of lysing reagent (Fig.11. 3.17).18 Applying the blood sample to the test card Fig. 4.17 Adding a blood sample to lysing reagent Fig. Immunological and serological techniques 345 11. 11. Fig. 11. When the reagent has been absorbed. 11. 11.

the fluorescent treponemal antibody-absorbed (FTA-Abs) test and the T. it shows up in many other conditions and disease states unrelated to treponemal infection.346 Manual of basic techniques for a health laboratory If the result is negative only the broken line is seen. pallidum and to nonpathogenic treponemes) of the normal bacterial flora of the oral or genital tract can also develop. The serum is then layered over a glass slide on which killed T. especially in the very early and very late stages. washed and overlaid with a fluorescent-labelled antihuman immunoglobulin antibody. FTA-Abs test The FTA-Abs test is used in the confirmation of syphilis. If the test result is positive the treponemes will fluoresce. as a rapid screening test for the following reasons: q q q There is no need for daily preparation of reagents. serum is diluted in a concentrated culture filtrate of Reiter treponemes to absorb any antibodies to nonpathogenic treponemes. Specific antibodies to treponemes (both to T. Immune responses to syphilis can be grouped into two categories: non-specific (or reaginic) and specific. Once positive this test remains positive for the life of the patient. and a special condition of maternal–fetal transmission termed congenital syphilis.10 Tests for syphilis infection Syphilis is caused by Treponema pallidum. Routine tests for syphilis include the rapid plasma reagin (RPR) test. Heat inactivation of serum is not required. Since the reaginic antibody lacks specificity. No microscope is required. A similar test for P.21 Clearing the lysed blood with washing reagent The non-specific reagin is of the IgM class and reacts with an alcoholic extract of beef heart known as cardiolipin (a phospholipid). It is not used as a screening test for syphilis because it does not detect reinfection and it is time-consuming and costly (a fluorescence microscope with a dark-field condenser is required). Remember that a . vivax is under development. pallidum organisms (Nichols strain) have been affixed. 11. and charcoal particles to allow the results of the reaction to be read without a microscope. Current studies indicate that the test has a sensitivity and a specificity of 86–95% when compared with standard light microscopy carried out by experienced technicians. latent and tertiary. The whole test takes less than 10 minutes. The slide is incubated. Principle RPR test The RPR test has replaced the Venereal Disease Research Laboratory (VDRL) test. 11. pallidum haemagglutination (TPHA) test. The RPR test can also be applied as a semi-quantitative test. Fig. These antibodies are of the IgG class and remain detectable throughout the life of the patient despite treatment. This indirect immunofluorescence technique is highly sensitive in all stages of syphilis. In the first step of the test. The RPR test uses the VDRL antigen modified with choline chloride to inactivate complement. The results of a test for syphilis must be interpreted according to the type(s) of test employed and the stage of the disease the patient has reached. secondary. In these cases false-positive reactions can occur. There are four stages of syphilis infection: primary.

Method 1. 11. 2. If the test result is positive the erythrocytes will form a smooth mat of agglutinated cells. The serum is then transferred to a microtitre plate and erythrocytes sensitized with killed T. 53). pallidum organisms (Nichols strain) are added.11. TPHA test The TPHA test is also used in the confirmation of syphilis. diluted serum is mixed with absorbing diluent containing non-pathogenic Reiter treponemes. . q q q q Weakly positive results may be due to: — very early infection. A negative result may mean one of the following: q q The infection is too recent to have produced detectable levels of antibodies. faulty technique or to the presence of other treponemal antibodies. The greatest value of the non-treponemal tests is in screening following therapy and in the detection of reinfection. The patient has not produced protective antibodies because of immunological tolerance. 0. weak-positive and strongly positive controls Sodium chloride. The technique is faulty. 75 mm ¥ 12 mm Test-tube rack Rotator RPR antigen Negative. Immunological and serological techniques 347 positive result from a screening test for syphilis may be due to other heterophile antibodies.10. Bring the test and control sera and RPR antigen to room temperature.85% solution (reagent no. 1 Note: The reagents for the RPR test should be stored at 2–6 °C in the refrigerator. The test is temporarily non-reactive because of treatment the patient is receiving. The above reagents are usually supplied as part of a test kit. In the first step of the test. — nonspecific immunological reactions.1 RPR test Materials and reagents1 q q q q q q q q q Test plates Disposable Pasteur pipettes Serological pipette Test-tubes. The test has been rendered temporarily non-reactive because the patient has consumed alcohol prior to testing. — incorrect technique. The disease is latent or inactive. — lessening of the activity of the disease after treatment. Dispense one drop of each of the test and control sera on to the test plates and spread carefully in the individual wells.

10. If necessary. (The recommended speed is between 95 and 105 rpm and this should be checked daily as part of quality control. Add 75 ml of the control erythrocytes to the wells in the first vertical row (1) and every other row (3. positive and negative control sera Distilled water. 11. 4. 11. 11.22 RPR test 3. absorbing diluent. Place the test plates on the rotator and rotate for 8 minutes at 100 rpm. Dilute the test and control sera 1:20 with absorbing diluent. The highest dilution of serum to give flocculation is the titre. 7. Prepare a twofold dilution of any positive sera and examine the dilutions as described in steps 2–5. Method 1. erythrocytes sensitized with T. micropipettes (with disposable tips). 3. Repeat the procedure with the remaining test sera. Examine the test plates for flocculation (Fig. 9 and 11).23). . 3 and 4 in Fig. q The reagents and controls should be reconstituted before use according to the manufacturer’s instructions. 11.23).22) and compare the reactions of the test sera with those of the controls.23). 6. pallidum.23). 5. use the adjacent wells (e. 11. unsensitized erythrocytes. as appropriate. 11.348 Manual of basic techniques for a health laboratory Fig. Add one drop of RPR antigen to each well of the test plates. 2. dispense 25 ml of the negative control serum into wells 1 and 2 of the first horizontal row of the microtitre plate (A in Fig. Dispense 25 ml of the first test serum into wells 1 and 2 of the third horizontal row of the microtitre plate (C in Fig. tilt the plates back and forth and rotate the plates carefully for 8 minutes at 80–85 rpm.g. 5. 4. Using a micropipette. 11.) If a mechanical rotator is not available. Dispense 25 ml of the positive control serum into wells 1 and 2 of the second horizontal row of the microtitre plate (B in Fig.2 TPHA test Materials and reagents q q q Test-tubes Test-tube rack Commercially available TPHA test kit containing microtitre plates. 5.

If the result is positive a smooth mat of agglutinated cells will be seen.11. Immunological and serological techniques 349 Fig. Place the plates carefully on a white background or a sintered glass plate illuminated from below or a viewing device that allows the sedimentation pattern to be seen from below through a mirror. . Note: The results should be interpreted according to the criteria provided by the manufacturer. 11. cover and leave to stand at room temperature for the time recommended by the manufacturer. or may even cover the entire base of the well. 8. If the result is doubtful (borderline) a button of non-agglutinated cells with a small hole in its centre will be seen. 8. with or without a very small hole in its centre. 10 and 12). The plates should be protected from vibration.23 Test plate for the TPHA test 6. Rotate the plates carefully. 6. radiant heat and direct sunlight. as appropriate. The cells may be surrounded by a red circle. Add 75 ml of the sensitized erythrocytes to the wells in the second vertical row (2) and every other row (4. 7. If the result is negative a compact red button of non-agglutinated cells will be seen.

5 g q.350 Manual of basic techniques for a health laboratory ANNEX Reagents and their preparation Order Reagents are listed in alphabetical order. 200 ml Label the bottle “ACETIC ACID 10% SOLUTION” and write the date. 1000 ml Chemical formulae In most cases the chemical formulae of the compounds used are given immediately after the English names: — sodium chloride (NaCl) — potassium hydroxide (KOH) — sulfuric acid (H2SO4) etc. Add enough water (q. 100 g/l (10%) solution (No. Warning: Glacial acetic acid is highly corrosive. For example: acetic acid brilliant cresyl blue carbol fuchsin hydrochloric acid sodium carbonate is under A is under B is under C is under H is under S Each reagent has a number which appears after the name (the numbers are given in the techniques).s.s. q. 2) Glacial acetic acid (CH3COOH) Distilled water 20 ml q. This can be useful when checking the label on the bottle. Acetic acid.s. = the quantity required to make up a certain volume For example: sodium chloride distilled water This means: Place 8. 350 .s. 50 g/l (5%) solution (No.5 g of sodium chloride in a volumetric flask.) to obtain a total volume of 1000 ml. Warning: Glacial acetic acid is highly corrosive. Acetic acid. 8. 200 ml Label the bottle “ACETIC ACID 5% SOLUTION” and write the date.s. 1) Glacial acetic acid (CH3COOH) Distilled water 20 ml q.

4) Acetone Absolute ethanol Distilled water 200 ml 475 ml 25 ml Mix the acetone. 500 ml Half fill a 500-ml flask with distilled water. ethanol and distilled water and transfer to a clean glass-stoppered bottle. The reagent will keep for at least 6 months at 2–8 °C.Annex.s. Mix well. Albert stain (No. Warning: Glacial acetic acid is highly corrosive. 5) Hydrochloric acid (HCl). Store at 2–8°C. Transfer the reagent to a brown bottle. Acid–ethanol for Ziehl–Neelsen stain (No.s. 7) Toluidine blue Malachite green Glacial acetic acid (CH3COOH) Ethanol (CH3CH2OH). 85% Cadmium sulfate Thiosemicarbazide Distilled water 44 ml 66 ml 1. and follow with the orthophosphoric acid. 200 ml Label the bottle “ACETIC ACID 50% SOLUTION” and write the date. Reagents and their preparation 351 Acetic acid. 96% Distilled water 0. Warning: Sulfuric acid is highly corrosive.15 g 0. Store at room temperature. Add the ethanol and make up the volume to 100 ml with distilled water. 500 g/l (50%) solution (No.6 g 50 mg q. . 100 ml Dissolve the glacial acetic acid in 30 ml of distilled water in a clean 100-ml bottle. stirring constantly. 3) Glacial acetic acid (CH3COOH) Distilled water 100 ml q. Label the bottle “ACID REAGENT” and write the date. Make up the volume to 500 ml with distilled water. Add the toluidine blue and malachite green and mix well.s. concentrated Ethanol (CH3CH2OH). Warning: Hydrochloric acid is highly corrosive.20 g 1 ml 2 ml q. Label the bottle “ALBERT STAIN” and write the date. add the sulfuric acid very slowly. 95% 3 ml 97 ml Label the bottle “ACID–ETHANOL FOR ZIEHL–NEELSEN STAIN” and write the date. Acetone–ethanol decolorizer for Gram stain (No. Warning: Glacial acetic acid is highly corrosive. Acid reagent (No. Continue mixing the solution and add the thiosemicarbazide and then the cadmium sulfate. 6) Concentrated sulfuric acid (H2SO4) Orthophosphoric acid (H3PO4). Label the bottle “ACETONE–ETHANOL DECOLORIZER” and write the date.

Stir using a glass rod until the crystals have completely dissolved. Add the agar and heat until the agar has completely dissolved. Fill a clean cuvette with distilled water. Quality control of alkaline haematin D reagent An alkaline haematin D standard solution (AHD standard) supplied by the central laboratory is used to test the quality of new batches of AHD reagent in peripherallevel laboratories. Check the quality of the solution (see below). Pipette 20 ml of AHD standard into a test-tube containing 3 ml of the freshly prepared AHD reagent (1:150 dilution). Store at room temperature (20–25°C). The AHD standard stock solution will keep for 8 months at 4–8°C. 1 (or equivalent) filter-paper. paying attention to accurate measurement of the constituents and the cleanliness of the glassware.0 g 1.2). pharmaceutical grade Sodium chloride (NaCl) Disodium hydrogen phosphate (Na2HPO4·2H2O) Potassium dihydrogen phosphate (KH2PO4) Sodium thioglycollate Calcium chloride (CaCl2). If a precipitate forms during storage. the reagent should be filtered before use. 1. If the haemoglobin values differ by more than 5 g/l.10 g 0. Replace the distilled water with AHD reagent.15 g 0. 5. Amies transport medium (No. 4. Filter the solution into a clean glass-stoppered reagent bottle. Add the charcoal.10 g 4. The haemoglobinometer or colorimeter should read zero. Place the cuvette in the cuvette chamber and adjust the haemoglobinometer or colorimeter to read zero at 540 nm wavelength.10 g 0. 2. 6.3. anhydrous Magnesium chloride (MgCl2) Agar Distilled water 10.20 g 0. Repeat the procedure using the previous batch of AHD reagent. Measure the haemoglobin concentration of the AHD standard (see section 9. Dispense into small tubes or bottles while stirring to keep the charcoal evenly suspended. 3. 9) Charcoal. Alkaline haematin D (AHD) reagent will keep for several months at 20–25°C. Note: Use filtered rainwater if distilled water is not available.0 g 3.00 g 1000 ml Suspend the mixture of salts in the distilled water. 8) Sodium hydroxide (NaOH) Triton X-100 (or equivalent) Distilled water 4g 25 g 1000 ml Dissolve the sodium hydroxide in the distilled water in a clean conical flask. using Whatman No. . discard the freshly prepared AHD reagent and prepare a new batch. Add the Triton X-100 (or equivalent) and mix well. Compare the results.352 Manual of basic techniques for a health laboratory Alkaline haematin D reagent (No. Label the bottle “ALKALINE HAEMATIN D REAGENT” and write the date.

Buffered glycerol saline (No. 12) Boric acid Distilled water 4. Label the bottle “BLANK REAGENT” and write the date. Transfer the solution to a glass-stoppered bottle. Blank reagent (No. 11) Trichloroacetic acid (CCl3COOH).4 g 100 ml Dissolve the dye and the trisodium citrate together in the sodium chloride solution.2 g 3. The reagent will keep for several months at 20–25°C. Transfer the solution to a glass-stoppered bottle. 100 ml Mix. 50 g/l (5%) solution (5 g in 100 ml of distilled water.1 g 1.s.s. 1000 ml Dissolve the copper sulfate crystals by heating in 100 ml of distilled water. 10) Copper sulfate (CuSO4·5H2O) Trisodium citrate (Na3C6H5O7·2H2O) Sodium carbonate (Na2CO3).0 g 0. Label the bottle “BORIC ACID SATURATED SOLUTION” and write the date. anhydrous Phenol red 4. stirring constantly. Store at room temperature (20–25°C).003 g . Benedict solution (No. 1000 ml Store in a glass-stoppered bottle. Dissolve the trisodium citrate and the sodium carbonate in about 800 ml of distilled water. Label the bottle “BRILLIANT CRESYL BLUE” and write the date. saturated solution (No. 53) 1. Warning: Trichloroacetic acid is highly corrosive.Annex. Label the bottle “BENEDICT SOLUTION” and write the date. anhydrous Distilled water 17. Make up the volume to 1000 ml with distilled water.85%) solution (No. Filter the solution obtained into a staining bottle. Amies transport medium is also available commercially.0 g 0. Brilliant cresyl blue (No. Label the tubes or bottles “AMIES TRANSPORT MEDIUM” and write the date.3 g 173 g 100 g q. see No. anhydrous Potassium dihydrogen phosphate (KH2PO4).8 g q. Reagents and their preparation 353 Sterilize by autoclaving at 120°C for 15 min. Boric acid. 14) Sodium chloride (NaCl) Dipotassium hydrogen phosphate (K2HPO4). 8. Add the copper sulfate solution slowly to the sodium carbonate/ trisodium citrate solution.5 g/l (0. Cool immediately in cold water to keep the charcoal evenly suspended. 62) 50 ml Distilled water q. 13) Brilliant cresyl blue Trisodium citrate (Na3C6H5O7·2H2O) Sodium chloride (NaCl).s.

1000 ml Dissolve the salts in the distilled water.2 700 ml 300 ml Dispense the solution into bijou bottles so that there is only a 2-cm gap between the top of the medium and the top of the bottles.0–7.2 (No.8 g 2.0 g 5. agar and distilled water to a clean 1000-ml beaker and mix. and tighten the caps. pH 7. 16) Solution A (saturated solution of basic fuchsin): Basic fuchsin Ethanol (CH3CH2OH).5 g 1. Check the pH using narrowrange pH papers. anhydrous Distilled water 3. Cary–Blair transport medium (No. anhydrous Sodium chloride (NaCl) Agar Distilled water 1. 95% Solution B (phenol aqueous solution. add 9 ml of freshly prepared aqueous calcium chloride (CaCl2).1 g q. Giemsa and Leishman stains.4. Warning: Phenol is highly corrosive and poisonous. Autoclave the vials containing the media for 15 minutes. 17) Sodium thioglycolate Disodium hydrogen phosphate (Na2HPO4). Label the bottles “BUFFERED GLYCEROL SALINE” and write the date. .2. Heat while mixing until the solution becomes clear. Label the vials “CARY–BLAIR TRANSPORT MEDIUM” and write the date. Buffered water. 50 g/l (5%)): Phenol (C6H5OH) Distilled water 10 g q. stirring well. Label the bottle “CARBOL FUCHSIN SOLUTION” and write the date. Transfer the solution to a glass-stoppered bottle.354 Manual of basic techniques for a health laboratory Distilled water Glycerol (C3H8O3) Final pH = 7. Dispense the solution in 7-ml volumes into previously rinsed and sterilized 9-ml screw-capped vials. cool. Label the bottle “BUFFERED WATER” and write the date. Transfer the resulting mixture to a glass-stoppered bottle.1 g 5.s. Disodium hydrogen phosphate (Na2HPO4·2H2O) Potassium dihydrogen phosphate (KH2PO4).0 ml Add the salts. it should be 7. Carbol fuchsin solution for Ziehl–Neelsen stain (No. 200 ml 3g 100 ml Mix 10 ml of solution A with 90 ml of solution B. 10 g/l (1%) solution. and adjust the pH to about 8.0 g 991. Cool to 50 °C. 15) Buffer solution for May–Grünwald.s.

Potassium dichromate (K2Cr2O7) Concentrated sulfuric acid (H2SO4) Distilled water 100 g 100 ml 1000 ml Dissolve the dichromate in the distilled water. modified Hucker (No. Add the haematoxylin and stir until it has dissolved. Label the bottle “DICHROMATE CLEANING SOLUTION” and write the date. For laboratories equipped with an accurate balance.0 g 8. MODIFIED HUCKER” and write the date. Store for 24 hours before use. The stain will keep for several months at 20–25°C. Mix well. Dichromate cleaning solution (No. Transfer the stain solution to a staining bottle. 18) Solution A Crystal violet Ethanol (CH3CH2OH).2 g 125 ml 410 ml Warm the ethanol by placing the beaker in a bowl of hot water. Allow the solution to cool. Drabkin diluting fluid (No. Transfer the solution to a glass-stoppered bottle.s. The acid must always be added to the water. use the solution as seldom as possible. then filter.8 g 80 ml 2. Label the bottle “DELAFIELD’S HAEMATOXYLIN STAIN” and write the date. 19) Haematoxylin Ammonium alum Potassium permanganate Absolute ethanol Distilled water 4. Warning: Since potassium dichromate and sulfuric acid are both corrosive and the mixture even more so.40 g 0. The instructions for its preparation are supplied by the manufacturer. 21) Drabkin diluting fluid can be prepared from commercially available reagent tablets. stirring constantly. 2000 ml . Drabkin diluting fluid can be prepared as follows: Potassium ferricyanide (K3Fe(CN)6) Potassium cyanide (KCN) Potassium dihydrogen phosphate (KH2PO4) Sterox SE (or equivalent) Distilled water 0. Delafield’s haematoxylin stain (No.10 g 0. Reagents and their preparation 355 Crystal violet.28 g 1 ml q. Dissolve the potassium permanganate in 10 ml of distilled water and add this solution to the stain solution. Add the ammonium alum to 400 ml of distilled water (warmed to 40°C) and stir until it has dissolved.0 g 0. Filter the stain solution into a staining bottle. 95% Solution B Ammonium oxylate ((NH4)2CO4·H2O) Distilled water 0. Add the solution to the filtered haematoxylin solution and mix well. Filter into a staining bottle.Annex. Label the bottle “CRYSTAL VIOLET. not the water to the acid.0 g 20 ml Mix solutions A and B. 20) For cleaning glassware. Add the acid gradually. Store at room temperature (20–25°C).

Add the remainder of the solution. 53) 2g q. When measured against water as blank in a spectrophotometer at a wavelength of 540 nm.04 ml of this solution into small containers marked to hold 2. anhydrous Distilled water 1. 22) Dipotassium ethylenediaminetetraacetate (potassium edetate) Distilled water 20 g q. EDTA dipotassium salt. heated to 80 °C 5g q. 100g/l (10%) solution (No. The reagent should be clear and pale yellow in colour. wash your hands thoroughly. Allow the anticoagulant to dry by leaving the containers overnight on a warm bench or in an incubator at 37 °C. anhydrous Potassium dihydrogen phosphate (KH2PO4). . Label the bottle “DRABKIN DILUTING FLUID” and write the date. Eosin. pipette 0.0 g 10. 24) Eosin Sodium chloride (NaCl). 10 g/l (1%) solution (No. discard. Filter when cool into a 1000-ml bottle. 100 ml Label the bottle “EOSIN 2% SOLUTION IN SALINE” and write the date. Preparation from original stains and chemicals Methylene blue (medicinal) Azur 1 Disodium hydrogen phosphate (Na2HPO4). If the reagent appears cloudy. Label the bottle “FIELD STAIN A” and write the date. Warning: Potassium cyanide is a highly poisonous chemical and should be used only by experienced chemists. 200 ml For use.s. Add the detergent and mix gently. 23) Eosin Distilled water 1g q. Label the bottle “FIELD STAIN A” and write the date.356 Manual of basic techniques for a health laboratory Dissolve the first three chemicals in the distilled water and mix.5 g q. When not in use it should be kept in a locked cupboard.s. 600 ml Mix until dissolved. 8. Add the stain powders and mix well.s.85%) solution (No.s.5 g/l (0.5 ml of blood. the absorbance should be zero.6 g 1. Eosin. Pour about half of the solution into a 1000-ml bottle containing a few glass beads.0 g 12. Mix well and filter into a clean 1000-ml bottle. 1000 ml Dissolve the two salts in the distilled water.s. Transfer the diluting fluid to a brown bottle. 100 ml Label the bottle “EOSIN 1% SOLUTION” and write the date. 20 g/l (2%) solution in saline (No. 25) Field stain A Preparation from prepared powders Field stain A powder Distilled water. After using the chemical. Field stain (No.

26) Sodium fluoride (NaF) Potassium oxalate (KCOOH) Distilled water 1. Reagents and their preparation 357 Field stain B Preparation from prepared powders Field stain B powder Distilled water. Formaldehyde saline (No. Fluoride oxalate anticoagulant (No. 10% solution (No. Filter into a clean 1000-ml bottle. 52).1 ml of the anticoagulant into small containers. they should be filtered every 2–3 days. anhydrous Potassium dihydrogen phosphate (KH2PO4). Undiluted. 1000 ml Dissolve the two salts in the distilled water. Warning: Both sodium fluoride and potassium oxalate are poisonous.s. 600 ml Mix until dissolved. After dilution. heated to 80 °C 4. Giemsa stain (No. Warning: Formaldehyde is corrosive and poisonous. anhydrous Distilled water 2.Annex.85%) solution (No.5 g/l (0. 100 ml For use.s. Add the eosin. 8. 50 g/l (5%) solution (No. Preparation from original stain and chemicals Eosin (yellow water-soluble) Disodium hydrogen phosphate (Na2HPO4). Label the bottle “FIELD STAIN B” and write the date.75 g 65 ml 35 ml . Warning: Formaldehyde is corrosive and poisonous.8 g q.0 g 12. at least 37% (formalin) Sodium chloride (NaCl). Filter when cool into a 1000-ml bottle. 53) 10 ml 90 ml Commercial formaldehyde solution is neutralized by adding a few drops of sodium carbonate. Pour into a 1000-ml bottle. marked to hold 2 ml of blood (or CSF). Test with pH indicator paper. Formaldehyde.0 g q.5 g q. at least 37% (formalin) Distilled water 100 ml 300 ml Transfer the solution to a glass-stoppered bottle.s. Label the bottle “FORMALDEHYDE SALINE” and write the date. Field stains can be used for as long as they give good results. 29) Powdered Giemsa stain Methanol (CH3OH) Glycerol (C3H8O3) 0. Label the bottle “FORMALDEHYDE 10% SOLUTION” and write the date. Mix until dissolved.0 g 10.2 g 6. Label the bottle “FIELD STAIN B” and write the date. 27) Neutral commercial formaldehyde (CH2O) solution. pipette 0. 28) Commercial formaldehyde (CH2O) solution.

(If it is difficult to dissolve. 500 ml Label the flask “GLUCOSE STOCK REFERENCE SOLUTION 100 MMOL/ L” and write the date.s. stand the flask in a bowl of hot water. 10.1% SOLUTION” and write the date. Allow to stand for at least 24 hours before use. Glucose working reference solutions (2. Benzoic acid. 1000 ml Measure 1000 ml of distilled water and heat to just below boiling. Keep in the refrigerator. Add distilled water to dissolve the chemical. anhydrous Benzoic acid.1%) solution Benzoic acid Distilled water 1g q. Warning: Glacial acetic acid is highly corrosive. Glucose reagents (No. 5. Keep at room temperature. Allow to cool. Transfer the solution to a 1000-ml glass-stoppered bottle. o-Toluidine reagent Thiourea Glacial acetic acid (CH3COOH) o-Toluidine 0. 20 and 25 ml of the stock reference solution into each of five 100-ml volumetric flasks. Transfer to a 500-ml flask and make up the volume to 500 ml with distilled water. Transfer to a beaker. Label the flask “TRICHLOROACETIC ACID 3% SOLUTION” and write the date. 10. 20 and 25 mmol/l) Allow the glucose stock reference solution to reach room temperature. Warning: Trichloroacetic acid is highly corrosive. 30 g/l (3%) solution Trichloroacetic acid (CCl3COOH) Distilled water 15 g q.s. pure. . Use a new bottle of frozen stock reference solution each time the working reference is prepared. 1 g/l (0. Label the bottle “GIEMSA STAIN” and write the date. 500 ml Weigh the acid out quickly. Glucose stock reference solution (100 mmol/l) Glucose. Carefully pipette 2. Shake the bottle three times a day for 4 consecutive days.5.358 Manual of basic techniques for a health laboratory Put the ingredients in a bottle containing glass beads and shake. Renew monthly. 5. 1 g/l (0. Add the benzoic acid and mix well until it is dissolved. Transfer the reagent to a brown bottle.75 g 470 ml 30 ml Dissolve the thiourea in the glacial acetic acid. Label the flasks as above and write the date.) Add the o-toluidine and mix well. Freeze in quantities of about 100 ml. since it is highly deliquescent.s. Make up to the mark with the benzoic acid solution and mix well.1%) solution 9g q.5. Store in a refrigerator. Filter into a staining bottle. Label the bottle “BENZOIC ACID 0. 30) Trichloroacetic acid. Label the bottle “O-TOLUIDINE REAGENT” and write the date.

Close the bottle tightly and keep it in the dark. Add the cotton blue and mix. lactic acid and glycerol to the distilled water. The stain is ready for use the following day. Prepare a stock solution of malachite green. Label the bottle “MALACHITE GREEN 1% SOLUTION” and write the date. 32) Hydrochloric acid (HCl). 1% stock solution Distilled water 100 ml 1 ml 100 ml Add the glycerol. Transfer the solution to a glassstoppered bottle. Warning: Hydrochloric acid is highly corrosive. 31) 1.s. grind the malachite green crystals to a powder.6 ml q. Label the bottle “HYDROCHLORIC ACID 0. drop by drop.01 mol/l solution (No. 1000 ml Rinse out a clean staining bottle with methanol.5 g q. 34) Leishman powder Methanol (CH3OH) 1. Add the acid. Lactophenol cotton blue mounting solution (No. Add a few clean dry glass beads. concentrated Distilled water 8. Label the bottle “LACTOPHENOL COTTON BLUE MOUNTING SOLUTION” and write the date. Label the bottle “GLYCEROL–MALACHITE GREEN SOLUTION” and write the date.01 MOL/L SOLUTION” and write the date. 33) Cotton blue (aniline blue) Phenol (C6H5OH) crystals Lactic acid (CH3CH(OH)COOH) Glycerol (C3H8O3) Distilled water 50 mg 20 mg 20 ml 40 ml 20 ml Add the phenol. 2. Isotonic saline See Sodium chloride. Prepare a working solution of glycerol–malachite green: Glycerol Malachite green. 1000 ml Measure out 500 ml of distilled water into a 1000-ml glass-stoppered bottle. Dissolve 1 g of the freshly prepared powder in 100 ml of distilled water and pour the solution into a dark bottle. Reagents and their preparation 359 Glycerol–malachite green solution (No. mix and dissolve by heating gently. Hydrochloric acid. Add the staining powder and methanol and mix well. Make up to 1000 ml with the rest of the distilled water. 0.Annex. Mix gently before use. Label the bottle “LEISHMAN STAIN” and write the date. Warning: Phenol is highly corrosive and poisonous. . Renew monthly. malachite green stock solution and distilled water to a 250-ml glass-stoppered bottle.s. Leishman stain (No. 1% solution: Malachite green Distilled water 1g 100 ml Using a pestle and mortar. It is important to prevent moisture from entering the stain during its preparation and storage.

Label the bottle “LOEFFLER METHYLENE BLUE” and write the date. a few millilitres at a time. Alternatively. 5 g/l (0. 38) May–Grünwald powder Methanol 5g q. ethanol and the remainder of the distilled water and mix well. Add the remainder of the distilled water and mix well. 200 g/l (20%) solution (No.1%) solution (No.5 g 30 ml 0. 35) Methylene blue Absolute ethanol (CH3CH2OH) Potassium hydroxide (KOH). 36) Iodine Potassium iodide (KI) Distilled water 1g 2g 300 ml Grind the dry iodine and potassium iodide in a mortar. mixing at intervals. Label the bottle “LUGOL IODINE 0. Label the bottle “LUGOL IODINE 0. measure 300 ml of distilled water in a cylinder. Label the bottle “MAY-GRÜNWALD STAIN” and write the date. Add the iodine and mix until dissolved. a few millilitres at a time.5%) solution (No. First dissolve the potassium iodide in about 30 ml of the distilled water. Store in a brown bottle. Add a few clean dry glass beads. First dissolve the potassium iodide in about 30 ml of the distilled water.s.s. . Alternatively. Add the staining powder and methanol. 37) Iodine Potassium iodide (KI) Distilled water 5g 10 g q. Lugol iodine.1% SOLUTION” and write the date.360 Manual of basic techniques for a health laboratory Loeffler methylene blue (No. and grind thoroughly after each addition until the iodine and iodide dissolve. and grind thoroughly after each addition until the iodine and iodide dissolve.s. The stain is improved by keeping for 1–2 weeks. May–Grünwald stain (No. Lugol iodine. 1 g/l (0. 100 ml Dissolve the methylene blue in 30 ml of distilled water and transfer the solution to a clean brown bottle. 45) Distilled water 0. Rinse the solution into an amber glass bottle with the remainder of the distilled water.1 ml q. Add distilled water. Mix well to dissolve the stain. Add distilled water. Rinse the solution into an amber glass bottle with the remainder of the distilled water.5% SOLUTION” and write the date. It is important to prevent moisture from entering the stain during its preparation and storage. Add the iodine and mix until dissolved. Store in a dark place at room temperatures (20–25°C). Add the remainder of the distilled water and mix well. 300 ml Grind the dry iodine and potassium iodide in a mortar. Add the potassium hydroxide. measure 300 ml of distilled water in a cylinder. 1000 ml Rinse out a clean 1000-ml bottle with methanol. Store in a brown bottle.

42) Phenol red crystals 0. anhydrous (NaH2PO4) Distilled water q. Store at room temperature (20–25°C). Add the distilled water and shake vigorously. pH 6. Prepare a stock solution of anhydrous sodium dihydrogen phosphate in a 1000ml volumetric flask: 13. Leave to stand for 1 day.1% SOLUTION” and write the date. 1 g/l (0. Pandy reagent (No. Check whether any phenol remains undissolved. Neutral red. 40) Neutral red Distilled water 1g q.8 (No.1 g Distilled water 10 ml Weigh out the phenol red crystals in a 20-ml beaker. 1000 ml . Filter the solution into a clean brown bottle. Reagents and their preparation 361 Methylene blue solution (No. Label the volumetric flask “ANHYDROUS SODIUM DIHYDROGEN PHOSPHATE” and write the date.s. Transfer the solution to a plastic dropper bottle. If so.s. Prepare a stock solution of disodium hydrogen phosphate in a 1000-ml volumetric flask: Disodium hydrogen phosphate. If anhydrous sodium dihydrogen phosphate is not available.8 g q. 2. Store at room temperature. Phosphate-buffered water. add a further 10 g and wait another day before filtering. Keep the stock solution in a refrigerator. 10 g/l (1%) solution (No. filter. Add the distilled water and stir until the crystals have dissolved. Label the bottle “METHYLENE BLUE SOLUTION” and write the date.1%) solution (No. Phenol red. 1000 ml Dissolve the neutral red in about 300 ml of distilled water in a clean 1000-ml bottle. (If all the phenol has dissolved. Label the bottle “PANDY REAGENT” and write the date. Label the bottle “NEUTRAL RED 0. 0. the solution can be prepared by dissolving 17.Annex. Make up the volume to 1000 ml with distilled water and mix well.3 g 100 ml Dissolve the methylene blue in the distilled water. 1000 ml Sterilize the stock solution by filtering it through a 0. 41) Phenol (C6H5OH) Distilled water 30 g 500 ml Put the phenol in a 1000-ml bottle. dihydrate (Na2HPO4·2H2O) Distilled water 17. 43) 1.) Warning: Phenol is highly corrosive and poisonous.6 g Sodium dihydrogen phosphate.01 mol/l.) (Pandy reagent is a saturated solution of phenol.2 g of sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O) in 1000 ml of distilled water. 39) Methylene blue Distilled water 0. Label the bottle “PHENOL RED 1% SOLUTION” and write the date.s.2-mm pore size filter.

0 19. stopper. pH of working solution Volume of stock solution (ml) NaH2PO4 6.0 91.5 94.0 g 52. because of the dangerous reagents involved.8 6. add the glycerol to the PVA powder and mix thoroughly with a glass rod until all particles appear coated with the glycerol.0 81. The pH should be as indicated in the table. Warning: Mercuric acid is highly poisonous.0 8.2 7.0 72.0 87. and mix by swirling. 0.362 Manual of basic techniques for a health laboratory Sterilize the stock solution by filtering it through a 0. 3. If the pH is too low.0 ml Dissolve the mercuric chloride in the ethanol in a stoppered flask (50 or 125 ml) by swirling at intervals.8 g of disodium hydrogen phosphate. if it is too high.2 56. stopper. Add the distilled water.3 Na2HPO4·2H2O 16.9 39.5 ml 5.5 5.9 7.8 g of disodium hydrogen phosphate.2-mm pore size filter. Mix the two stock solutions in the amounts shown in the table below to obtain 100 ml of buffered water. Glacial acetic acid is highly corrosive. 32). Keep the stock solution in a refrigerator.0 83.9 13.0 7.5 ml In a small beaker.01 mol/l solution (No. 95% Glacial acetic acid 1. PVA mixture Glycerol PVA powder (low viscosity) Distilled water 1. heptahydrate (Na2HPO4·7H2O) or 35. Scrape the mixture into a 125-ml flask.7 Polyvinyl alcohol (PVA) fixative (No. adjust it with sodium hydroxide (NaOH).01 mol/l solution (No. Add the acetic acid.8 8.1 61.8 25.5 6. Label the volumetric flask “DISODIUM HYDROGEN PHOSPHATE STOCK SOLUTION” and write the date. dodecahydrate (Na2HPO4·12H2O).0 28.0 49. the solution can be prepared by dissolving 26. and leave at room temperature (20–25°C) for 3 hours or overnight. If disodium hydrogen phosphate dihydrate is not available.4 7.0 50. Modified Schaudinn fixative Mercuric chloride crystals (HgCl2) Ethanol. 54). Swirl the mixture occasionally to mix. adjust it with hydrochloric acid (HCl).8 43. Label the flask “PVA MIXTURE” and write the date.6 7. 44) Note: This should be prepared at an intermediate level laboratory.5 g 31.2 75.4 6. . Label the flask “MODIFIED SCHAUDINN FIXATIVE” and write the date. 0.0 ml 5.

200 g/l (20%) solution (No. 53) 1g 100 ml .s. restopper and swirl to mix. Label the bottle “PVA FIXATIVE” and write the date. Transfer the solution to a glass-stoppered bottle. Prepare a working solution in a glass-stoppered bottle: Stock solution Distilled water 10 ml 90 ml Label the bottle “SAFRANINE WORKING SOLUTION” and write the date. Store the PVA fixative in a screw-cap or glass-stoppered bottle. 40 g/l (4%) solution (No. Adjust the heat to maintain this temperature range. 8. 45) Potassium hydroxide (KOH) pellets Distilled water 20 g q. 100 ml Label the volumetric flask “POTASSIUM HYDROXIDE 20% SOLUTION” and write the date. swirling frequently.5%) solution (No. to allow bubbles to escape. Prepare a stock solution: Safranine O Ethanol (CH3CH2OH). Safranine solution (No. 3. Make up the volume to 1000 ml with distilled water. 1000 ml Dissolve the potassium permanganate in 300 ml of distilled water in a 1000-ml volumetric flask. 5.5 g/l (8. Place the loosely stoppered flask containing the PVA mixture in the water-bath for about 10 minutes. 46) Potassium permanganate (KMnO4) Distilled water 40 g q.Annex. and to clear the solution. Potassium permanganate. 48) Saponin Sodium chloride (NaCl). Label the volumetric flask “POTASSIUM PERMANGANATE 4% SOLUTION” and write the date.s.s. Continue to swirl the mixture in the water-bath for 2–3 minutes to dissolve the remainder of the PVA. PVA fixative working solution 1. Saponin. There are many grades of PVA powder on the market. but the grades with high hydrolysis and low or medium viscosity are most satisfactory for preparing PVA fixative. 47) 1. 2.5 g q. Store in the dark. Reagents and their preparation 363 PVA powder and PVA fixative solutions are available from several commercial sources. Heat a water-bath (or large beaker of water) to 70–75°C. 10 g/l (1%) solution (No. pour in the modified Schaudinn fixative solution. Label the bottle “SAFRANINE STOCK SOLUTION” and write the date. 4. 95% 2. 100 ml Mix until all the safranine has dissolved. Remove the flask from the water-bath and allow it to cool. Warning: Potassium hydroxide is corrosive. When the PVA powder appears to be mostly dissolved. 2. The fixative will keep for 6–12 months. Potassium hydroxide.

1 g q. Sodium carbonate. Sodium carbonate.364 Manual of basic techniques for a health laboratory Add the sodium chloride solution to a glass bottle. 2 gl/l (0. Label the bottle “1% SAPONIN IN SALINE” and write the date.85% SOLUTION” and write the date. 0.7% SOLUTION” and write the date.s.01 mol/l solution (No. 51) Sodium carbonate (Na2CO3). 1000 ml Label the volumetric flask “SODIUM CHLORIDE 0.s. Sodium bicarbonate. 100 ml Label the volumetric flask “SODIUM CARBONATE 5% SOLUTION” and write the date.2%) solution (No. 17 g/l (1. 53) Sodium chloride (NaCl) Distilled water 8. Sodium citrate See Trisodium citrate.85%) solution (isotonic saline) (No.7%) solution (No. 20 g/l (2%) solution (No. Sodium hydrogen carbonate See Sodium bicarbonate. Sodium hydroxide. 52) Sodium carbonate (Na2CO3). 100 ml Label the volumetric flask “SODIUM BICARBONATE 2% SOLUTION” and write the date. 100 ml . 100 ml Label the volumetric flask “SODIUM CARBONATE 0. 49) Silver nitrate (AgNO3) Distilled water 5. Warning: Silver nitrate is caustic. anhydrous (or an equivalent quantity of one of the hydrates) Distilled water 2g q. 300 ml Mix until all the silver nitrate has dissolved.s. Add the saponin. 8.2% SOLUTION” and write the date. 54) Sodium hydroxide (NaOH) pellets Distilled water 3g q. and heat until it has completely dissolved.5 g/l (0. 50) Sodium bicarbonate (NaHCO3) Distilled water 2g q. anhydrous (or an equivalent quantity of one of the hydrates) Distilled water 5g q.5 g q.s.s. mix. 50 g/l (5%) solution (No. Sodium chloride. Silver nitrate.s. Label the bottle “SILVER NITRATE 1.

Stir until dissolved and add 10 g of neutral charcoal powder. 1 : 1000 Formaldehyde. anhydrous Sodium dihydrogen phosphate (NaH2PO4).15 g 0.s. 0.01 MOL/L SOLUTION” and write the date. 10 g/l (1%) aqueous solution (freshly prepared) Magnesium chloride (MgCl2). 30 g/l (3%) solution (No. Sodium metabisulfite.3 1. 3. Reagents and their preparation 365 Label the volumetric flask “SODIUM HYDROXIDE 0. 10% solution (No.s.s.20 g 1. Dispense 5–6 ml of medium per 13 mm ¥ 10 mm screw-capped tube (avoid crushing).Annex. Store in the refrigerator. Autoclave at 121 °C for 20 minutes. Warning: Sodium hydroxide is corrosive.00 g 1000 ml Sulfosalicylic acid. Label the tubes “STUART TRANSPORT MEDIUM. anhydrous Sodium thioglycolate Calcium chloride (CaCl2). 58) 1. Label the volumetric flask “SODIUM METABISULFITE 2% SOLUTION” and write the date. 56) Agar Distilled water Heat until dissolved and add while hot: Sodium chloride (NaCl) Potassium chloride (KCl) Disodium hydrogen phosphate (Na2HPO4). 57) Sulfosalicylic acid Distilled water 3g q. MODIFIED” and write the date. 28) Glycerol Distilled water 200 ml 25 ml 5 ml q. 2. modified (No. 3. 20 g/l (2%) solution (No.00 g 0.20 g 1. 55) Sodium metabisulfite (Na2S2O5) Distilled water Make up freshly for use. 250 ml . 25 ml Stuart transport medium. Invert the tubes before the medium solidifies in order to distribute the charcoal uniformly. 100 ml Label the volumetric flask “SULFOSALICYLIC ACID 3% SOLUTION” and write the date.5 g q. TIF (thiomersal–iodine–formaldehyde) fixative (No. 10 g/l (1%) aqueous solution Final pH: 7.00 g 10 ml 10 ml 4. Prepare a stock solution: Tincture of thiomersal.

2%) solution (No. . Transfer the solution to a 200-ml volumetric flask and make up the volume to 200 ml with distilled water.s. Warning: Trichloroacetic acid is highly corrosive. 62) Trichloroacetic acid. Urea reagents (No. Trisodium citrate. 61) Glacial acetic acid (CH3COOH) Methylene blue solution (No. 39) Distilled water 4 ml 10 drops q. 100 ml Keep in the refrigerator. Transfer to a beaker. 60) This is used as an anticoagulant.s. Label the bottle “THIOMERSAL– FORMALDEHYDE STOCK SOLUTION” and write the date. On the day of use. 50 g/l (5%) solution (No. 200 ml Dissolve the glacial acetic acid in 100 ml of the distilled water.5 g/l (0.s.2 g q.s. 2g q.2% SOLUTION” and write the date. Label the volumetric flask “TRISODIUM CITRATE 3.4 ml 0. Türk solution (No. 8. 20 g/l (2%) solution in saline (No. 100 ml Trisodium citrate. Label the volumetric flask “TRICHLOROACETIC ACID 5% SOLUTION” and write the date. dihydrate (Na3C6H5O7·2H2O) Sodium chloride. mix: Stock thiomersal solution Lugol iodine. Label the volumetric flask “TURK SOLUTION” and write the date. 2. Label the volumetric flask “TRISODIUM CITRATE 2% SOLUTION IN SALINE” and write the date. Transfer the mixture to a 200-ml volumetric flask and make up the volume to 200 ml with distilled water. Warning: Formaldehyde is corrosive and poisonous.366 Manual of basic techniques for a health laboratory Transfer the stock solution to a brown bottle. Add 100 ml of distilled water and mix to dissolve the chemical. 50 g/l (5%) solution Trichloroacetic acid (CCl3COOH) Distilled water 10 g q. Use 1 ml of the solution per 4 ml of blood. The stock solution will keep for up to 3 months.6 ml Trisodium citrate. it is highly deliquescent. 200 ml Weigh the acid out quickly. 59) Trisodium citrate.85%) solution (No. 37) 9. 32 g/l (3. Warning: Glacial acetic acid is highly corrosive. anhydrous (Na3C6H5O7) (or an equivalent quantity of either the dihydrate or the pentahydrate) Distilled water 3. Add the methylene blue solution and mix. 53) Keep in the refrigerator.

10 mmol/l Urea stock reference solution Benzoic acid (C7H6O2). The staining properties of Wayson stain improve with age. Urea stock reference solution. Warning: Phenol is corrosive. The solution will keep for at least 6 months at 2–8 °C.s. Label the flask “UREA STOCK REFERENCE SOLUTION 125 MMOL/L” and write the date. Colour reagent Acid reagent (No. 125 mmol/l Urea Benzoic acid. The reagent must be prepared daily. . Urea working reference solution. 1 g/l (0. 500 ml Label the volumetric flask “DIACETYL MONOXIME STOCK SOLUTION” and write the date. Solution B (phenol. Label the volumetric flask “UREA WORKING REFERENCE SOLUTION 10 MMOL/L” and write the date.1%) solution (see No.1%) solution (see No.Annex.3-butanedione monoxime) Distilled water 2g q. Make the stain in large quantities and dispense it in small amounts in to dark bottles for future use. Label the flask “COLOUR REAGENT”. Store in a refrigerator. 100 ml Mix the solutions well in a 100-ml volumetric flask. 1 g/l (0.s. The solution will keep for several months at 2–8 °C.s. Label the bottles “WAYSON STAIN” and write the date. 100 ml Dissolve the urea in about 20 ml of the benzoic acid solution in a 100-ml volumetric flask. Wayson stain (No. 50 g/l (5%) solution): Phenol (C6H5OH) Distilled water 100 g 2000 ml 7g 100 ml 2g 100 ml Add solution A to solution B. 6) Diacetyl monoxime reagent 50 ml 50 ml Mix the acid reagent and stock solution in a 100-ml stoppered flask. Reagents and their preparation 367 Diacetyl monoxime stock solution Diacetyl monoxime (also called 2. 30) 750 mg q. 63) Solution A1: Basic fuchsin Absolute methanol (CH3OH) Solution A2: Methylene blue Absolute methanol (CH3OH) Combine the two solutions to give solution A. 30) 8 ml q. The quantities shown above are sufficient for 33 measurements. Make up the volume to 100 ml with benzoic acid solution.

0 g 1. add 5 ml of glacial acetic acid to the solution. Put 0. Leave to cool and stand. 100 ml Just before use. This fixative should be made up by fully qualified and experienced technicians only. 66) Potassium dichromate (K2Cr2O7) Mercuric chloride (HgCl2) Sodium sulfate (Na2SO4) Distilled water 2.368 Manual of basic techniques for a health laboratory Willis solution (No. If it has all dissolved add a further 50 g. place them in an incubator at 37 °C. 65) Ammonium oxalate (NH4)2C2O4·H2O) Potassium oxalate (K2C2O4·H2O) Distilled water 1.2 g 0. Leave the open bottles to dry at room temperature or. Label the volumetric flask “WINTROBE SOLUTION” and write the date.s. Zenker fixative (No.8 g q. Label the volumetric flask “ZENKER FIXATIVE” and write the date. Label the bottle “WILLIS SOLUTION” and write the date.5 g 5. 64) This is a saturated solution of sodium chloride. Sodium chloride (NaCl) Distilled water 125 g 500 ml Dissolve the sodium chloride by heating the mixture to boiling point. Warning: Glacial acetic acid is highly corrosive and mercuric chloride is highly poisonous.s. Wintrobe solution (No. Dissolve the three salts in 50 ml of distilled water in a 100-ml volumetric flask. preferably. Filter into a corked bottle.5 ml of this mixture in each 5-ml bottle used for the collection of blood.0 g q. 100 ml Dissolve the two salts in 50 ml of distilled water in a 100-ml volumetric flask. . Make up the volume to 100 ml with distilled water. Make up the volume to 100 ml with distilled water. Check that some of the salt remains undissolved.

204 African trypanosomiasis 182–192. 217 urine specimens 240. 348–349 Anti-streptolysin O test (ASOT) 336–338 Applicators. 189–192 AHD. 201–202. 133. see Anti-streptolysin O test Aspiration body cavity fluids 218–220 buboes 218. 352 Alkalis corrosive injuries from 99–100 handling precautions 97 Allen & Ridley sedimentation technique 153–154. 128. 184–187. 292. 320 “Actinomycetes”. 221–223 smears 199–201 sputum/throat specimens 204–207. 66–69. 215–216 Bacteriological index (BI) 223–224 Bacteriological tests (see also Bacteria) equipment 37–38 laboratory registers 47. 259–260. 204 stool specimens 216–218. 330–331. 219 Bacteria 197–224 anthrax 220 body cavity fluids 218–220 diarrhoeal diseases 105 leprosy 220–224.Index Note: Page numbers in bold refer to main entries. see Alkaline haematin D AIDS. 336–349. 199–204. 117. 309 Anthrax (Bacillus anthracis) 204. immunology 330–331. 158 Anisocytosis 309. 113–115 pathogenicity 111 Anaemias 284. 133. 219 lymph nodes 183–185. 86–87. distilled water 25. 150 eggs 126. 116. 343–344 Autoclaves 33. 357 Antigens 329–330 antibody interactions 330 immunochemical techniques 330–335 tests 331–336. 333–334. 344–346. 205–207 staining techniques 199–201. 336. 123 motile forms 111–114. 336. 119–121. 313 abnormal erythrocytes 305–310. polymorphonuclear 310. 310 Basophils. disinfection 85 Alembics. 306–310 sickle-cell 306. 293. 25 Alkaline haematin D (AHD) 276–279. wooden 36 Ascaris lumbricoides (roundworm) 106 adults 146. 351 Albumin. 315 Analytical balances 32. 127–128. 314–316. 311. 69 Ancylostoma duodenale (hookworm) 106 adults 148. 287–288. 152 ASOT. 348–349 Anticoagulants 42–43. 260. 249–254. 50 Balances. 154 Amies transport medium 352–353 Amoebae 105 cysts 118–120. 133. identification 200. 353 Benzoic acid 358 369 . 339 CATT 188–192. 219 Antibodies 328–329. 184–185 Assays. 344–346. 121 Base units. 68–69. 204. 266. 201. urine 237 Alcohols. 86–88 Bacillus anthracis 204. 250–253 urogenital specimens 209–211. see Acquired immunodeficiency syndrome Albert stain 201. 152 larvae 157. 307. 236–237. SI 3 Basophilic staining 305 Basophilic stippling. erythrocytes 309. 263 Agglutination techniques 333–334. 147 eggs 126. italicized page numbers refer to illustrations. 307. 311 Batteries. Acids corrosive injuries from 98–99 handling precautions 97 Acquired immunodeficiency syndrome (AIDS) (see also Human immunodeficiency virus) 313. 127. 67–69 Balantidium coli 105. 204. 210–211. electrical supply 14–15 Battery-operated centrifuges 71 Benedict solution 236. 336–349. laboratory 32. 121. 308–310. 329 antigen interactions 330 immunochemical techniques 330–335 tests 331–336.

187. 159. 258–259. 286 dispatch 92 equipment 42–43. 323–324 parasites 159–160. 295–296 Blood cells 265–266. 127 Bleaches. urine 245. 286–287 haemoglobin concentration 7. 165. 307 urine specimens 241–243. 172–182. 317–318 thrombocyte number concentration 7. 167. 280–283. 320 malaria parasites 180–182 reticulocyte number concentration/ fraction 6. 166. 173 Plasmodium spp. 193–194. 265–266 plasma 312. 281. 166. 278. 166–196 microfilariae 159. laboratory accidents 100–101 Butane gas burners 97 Cables. 193–194 thin blood films 175. electrical 19 Cabot ring bodies 309. 75 Calibration colorimeters 277. 151 Blood specimens chemistry registers 47. 310 Calcium crystals. 241–243 Centrifugation 69. 354 Casts. 276.) 106. 245. 293–294 erythrocyte volume fraction 6. 280–282. 159–160. 166–168 . measurement 295–296. household 84–85 Bleeding time. 178. 243–244. see Bacteriological index Bilirubin crystals. 169. 306–314 urea concentrations 7. 288–290. 330–335 Biopsy specimens 93. 279. 173 Bubonic plague (Yersinia pestis) 203–204. 324 spectrophotometers 272–274. urine 248. 313 target 306. 247 Calcium hydroxide 85 Calcium hypochlorite 84–85 Calculations (see also Measurement) blood glucose concentration 7. 272–273 leukocyte number concentration 6. 77 Burns. 193–194 Trypanosoma spp. 189–192 Cary–Blair transport medium 216–217. 241–243. 244 Blood flukes (Schistosoma spp. 219 Buffered glycerol saline 217–218. 248 Binding tests. 182–194. 267–270. 354. 158 urine specimens 234. 49 collection 102. 142–143 Cells blood 125. see Card agglutination trypanosomiasis test Cavity body fluids. 318–319. 353– 354 Buffered water 29–31. specimens 218–219 Cellophane faecal thick smear technique 141–143. 311. 218. 164. 166. 256–259 disinfection 84–85 stool specimens 157–159. 185. 280–281. 125. 265–266. 325–327 Boric acid 353 Bottles 36–37 Brilliant cresyl blue 316–317. 246. 178 protozoa 172–194. 31. 261 identification 200. 261. 163–172. 160–163. 200 vaginal discharge 215 Capillary blood bleeding time 295–296. 241–242. 246. 265–266 clotting 266. 279 Campylobacter spp. 43 glucose concentrations 322–325. 300–304. 299–314. 258–259. 267–270. 287–288 erythrocyte sedimentation rate 292–295. 175. 321 urine protein 238–239 Calibrated dropping pipettes 75. 297–298 CSF specimens 256–259. immunology 330–335. 281 glucose concentration 325 Carbol fuchsin solution 354 Card agglutination trypanosomiasis test (CATT) 188–192. 171. 297–298. 271–279. 161–163 Blank reagent 353 Blastocystis hominis (yeast) 124–125. 312 polymorphonuclear 310–311. 324–325 blood urea concentration 327 erythrocyte number concentration 6. 319–321. 288–292 leukocyte type number fraction 6. 361–362 Bunsen burners 35 Burettes 77. 187. 266. 187. 95–96. 150–151. 353 Broken glass injuries 101 Brugia spp. 171. 185–187 test-tube cleaning 82 thick blood films 173–182. 216 Candida albicans CSF 257. urine 240. 272–273. 313. 243–244 Catheters 233 CATT.370 Index BI. 295–296 collection 164. 279–287.

261. 134 Clotting blood 266. 288. differential 32 Counting chambers 37 Fuchs–Rosenthal 258–259. 50 leukocyte number concentration/ fraction 6. 247 Cysts hyatid 151. 42–44 Corrosive injuries. 64–65 reusable syringes/needles 81 specimen containers 81–82. 60–61. 271–274 AHD method 276–277 calibration 277–278. 260 Chagas disease 192–194. 263. modified Hucker 355 CSF. 121 motile forms 111. 37 Cerebrospinal fluid (CSF) 255–264. urine 247. 245–248. 240. 134. 82 specimen dispatch 92 trypanosomes 259–260. 288–292 measurement 4–6 reticulocyte number 6. 297–298. 321 Condensers. microscopes 56. 265. 69–72. urine 236. 124– 125 stool examination 108. 210 Chemical formulae. 81 Cresols 83 Cresyl blue. 245–248 Crystal violet. 82 “sharps” 96 specimens 42–44. 130. 125 . 287–288 haemoglobin 7. 207 staining techniques 201. 258–259 Neubauer 259. 78–82 centrifuges 83 glassware 77–81. 202–203 Crystals. 122. 264 Mycobacterium leprae 223 urine 254 watery stools 216 Cutaneous leishmaniasis 195–196 Cuts. 56. 288–291. 266. 290 Counts (see also Number concentrations. syphilitic 210. 261 Cryptosporidium spp. 151 protozoa 118–125. 323–324 blood urea 325–327 CSF glucose 26l–262 erythrocyte number 6. CSF 257. 207. 258–259 meningitis 260–261. Number fractions) parasite eggs 251 sperm 214–215 Coverslips. reagents 350 Chemicals. 124 Colorimeters 32. 109 yeast 124–125. 255–264 blood in 256–259. storage 45 Children (see also Infants. 256–259 glucose concentration 7. 297–298 CSF 257 Coccidia 122. see Brilliant cresyl blue Cryptococcus neoformans. 284. 152–157 Concentrations blood glucose 322–325. slides 80. laboratory accidents 101 Cystine crystals. 278. 316–319 thrombocyte number 7. 121. acids/alkalis 98–100 Corynebacterium diphtheriae (see also Diphtheria) specimen dispatch 207. 284–285 leukocyte number 6. 261 leukocyte number concentration 291 leukocyte type number fraction 320 normal haemoglobin 275 normal thrombocytes 321 reticulocyte number concentration 318 venous blood collection 267 Chilomastix mesnili 115–116. reporting 52 Concentration techniques. 123–124. 124. 261–262 laboratory registers 47. 80. 117 pathogenicity 111 Cleaning 77–85. 58. parasites 152–156. 266. 324 Colour reagent 367 Communicable diseases. 69–73 balancing 72. 82 Clonorchis sinensis (Chinese liver fluke) 106. 134 Chloramine 85 Cholesterol crystals. 193–194 erythrocyte number concentration 287 erythrocyte volume fraction 284 Haemophilus influenzae 261. urine 247. 248 Chromatoid bodies 122 Ciliates cysts 121. 263–264. 193–194 Chancre. 279. 260–261 protein concentration 262. 262 specimen containers 44.Index 371 Centrifuges 32. 117. 60 Confidentiality 2 Containers cleaning 81–82. 288–290. 201–202 Counters. 121 Chinese liver fluke (Clonorchis sinensis) 106. 72 cleaning and maintenance 83 tubes 34. 119–122. 134. 130. 78–81 incubators 83 laboratory balances 66–67 microscopes 65. see Cerebrospinal fluid Cultures dispatch 206–207. Neonates) Chagas disease 192–194. 116. 271–279.

57. 144–146 tapeworms 148. 355–356 Drainage. 272–275 Diphtheria (see also Corynebacterium diphtheriae) 201. 226 EDTA dipotassium salt 356 Effusions. 211. 16 Duke method. 239. 37 Dropping pipettes. 211. 61. 216 Decontamination blood spills 84 laboratory accidents 101 microscopes 65 Definitions mass 3 mole 3 quantity 2 weight 3 Deionizers 33 Delafield’s haematoxylin stain 355 Demineralized water 27–29. 308–309 . 344 HIV 342. see Enzyme-linked immunosorbent assay Elliptocytes 308. identification 145. calibrated 75. water 24. 252. 295–296 Dysentery amoebae (Entamoeba histolytica) 113. 145 Dilutions AHD reference solution 277 Drabkin fluid 271–275. 152 adults 152 eggs 130. 159 Disinfectants (see also Cleaning. 149 eggs 130. 113–114 Dichromate cleaning solution 355 Dicrocoelium spp. 264 sputum 205. 73–77 Disposable materials 90–91. 134. 152–153 sputum/throat specimens 206–207 urine specimens 248. 37 Diabetes 236. (lancet fluke) 106. 355–356 haemiglobincyanide reference solution 272–273. 28–29. microscopes 56. 135 Direct examination CSF 256–257. leprosy 221. 201–202. 25–26 Distributors. 158 parasites and other matter 144–146. 249–251 Electric centrifuges 70–72. SI 3–5 Dessicators 34. 256–257 semen 212 stools 107. Sterilization) 85–90 Disodium hydrogen phosphate 361–362 Dispatch specimens 91–96. 58. disinfection 84 Drop bottles 35. 69 Dispensing. 342 urine 234. 90–91 Disposal laboratory waste 90–91 specimens 97 Distilled water 24–27. 107 urine 234 Dirofilaria spp. 216–218 throat 207. bleeding time 295–296. 129–144. plumbing 22–23. 345–346 hepatitis B 344. see Leukocyte type number fraction Digested meat fibres. 151 Ectothrix 226. 322 Diacetyl monoxime blood urea concentration 325–327 stock solution 367 Diaphragms. 209 Dispensary balances 69. sterilization 90. 136. 120 Differential counters 32 Differential diagnoses Entamoeba histolytica/Escherichia coli 114 larvae 157. 22–23 Drinking-water. 134 Dientamoeba fragilis 114.372 Index Dark-field microscopy 64. 253 Diphyllobothrium latum (fish tapeworm) 106. 207 urogenital 209. 131. liquids 73–77. 114–115. 136 Dipstick tests falciparum malaria 344–346. 75 Dry heat. 113–114 Ear lesions. 253–254 Dipylidium caninum (tapeworm) 106 adults 148. 33 Derived units. 206 stool 109–110. 93–94 CSF 263–264. 135. 221–223 Echinococcus granulosus (hydatid cyst) 151. 24 Drabkin diluting fluid 271–275. 149 Differential leukocyte counts. examination 218–219 Eggs helminths 126–144. 61 Diarrhoeal diseases (see also Intestinal parasites) common causes 105–106 dysentery amoebae 113. 89–90 Dual-voltage electrical equipment 16. 70–72 Electricity 12–20 equipment 15–17 equipment failure 17–20 meters 16 shocks from 101 supply sources 12–15 Electronic charge regulators 14 ELISA.

193–194 thin blood 173–179. precautions 97 Flasks 34.) liver. 356 Epithelial casts. 187. see Erythrocytes. 120 hartmanni 114. 138. 209. 113–114. 39–44 water demineralizers 27–29. 304. 169. 300–304. 115–116 pathogenicity 111 Flaming. 150–151 Fluorescent treponemal antibodyabsorbed (FTA-Abs) test. 250–251. 222 Faeces. 368 Flagellates cysts 121.Index 373 Endolimax nanus 114. 299–314. 244 Equipment 1–2 centrifuges 70–73. 115–116. 138 Fasciolopsis buski 106. 199 Fixatives 95. 341–342. electricity 17 First aid 98–101. 37 Expiry dates. 311. 362–363. 135–137 Enzyme immunoassay 331. transmission routes 106 stools 150–151. 106. 61 Eyes acid/alkali splashes in 99. 286–287 ESR. 99 Fish tapeworm (Diphyllobothrium latum) 106. 152–153 Flukes (see also Schistosoma spp. urine 243. parasite detection 168–170. 318 malaria infected 179 measurement 6–7 nucleated 292. see Flukes Flotation techniques. 241. 310. 147 eggs 126. parasite detection 152–153. 120 Endothrix 226. 38 Foreign substances. 134 Fixation biopsy specimens 95–96 smears 197. 244 Female patients. 343–344 Eosinophils. 165. 215 Fibrinogen. 307 abnormal 306–310. 187. sterilization 90. electrical 19. 193–194. 45 Erythrocytes 265. 166–167. 135–137. 265. microscopes 56. 120. 28–29 Erlenmeyer flasks 34. 306. 123 motile forms 111. 118. 36–37 Flatworms. 172 skin infection 160–163. 315 thin blood films 305–310. 110. 78–82 electrical 15–20. 284. 155–156 . 16–19 first-aid 98 laboratory 32–46. 306–314 Filter funnels 34 Filters microscopes 57 water 24. 306–310 Cabot ring bodies 309. 34–35. examination 218–220 Eyepieces. 162–163 blood infection 163–172. urine 244. 70–73 cleaning 77–90. 265. 245 Formaldehyde 357 stool preservation 109. 171–172 geographical distribution 160. 311 Eosin solution 117–118. 304–305 Filariae 159–172. reagents 46 Extension leads. 110 Formaldehyde–detergent sedimentation technique 154–156. 178–181. 138. 310 number concentration 6. 161–163 Filariasis. urine 243. 128. 121. 120 Enterobius vermicularis (pinworm) 106 adults 146. 287–288 sedimentation rate (ESR) 292–295. electrical equipment 17–20 False casts. 250–251 Fire risk. 113–114. 36–37. 138 Fatty casts. polymorphonuclear 310. 24 Filtration. 199. urogenital specimens 208. 114. 226 Entamoeba coli 113. urine specimens 244–245. see Stools Failure. 113. 175–176. 279–287. 152 eggs 128. 241–242 volume fraction 6. smear staining 201 Fasciola spp. 120. 310 CSF 257–258 haemoglobin relationship 284–285 immature (reticulocytes) 316–319. 128. 134. 331 Enzyme-linked immunosorbent assay (ELISA) 331. 90 Flammable liquids. sedimentation rate Estimation. 118. 128. 120 histolytica 106. 36. see Filariae Films (see also Slide preparation) thick blood 173–182. 19 Exudates. deficiency 298 Field stains 356–357 faecal trophozoites 117 malaria parasites 177–178 thin blood films 299. 244 False positives/negatives. 114. 61. see Calculation Evaporating dishes 34. syphilis 346–347 Fluoride oxalate anticoagulant 357 Forceps 35. 175. 100 filarial infection 160 Facial lesions. 99. 293–294 sickle-cell anaemia 314–316. 306–310 urine 240. 280–283. leprosy 221–223.

152. 138–139. 228–229 CSF 257. 284–285 H bodies 319 Haemoglobinometers.374 Index Formaldehyde–ether sedimentation technique 153–154. 299–300. 78–81 disinfection 84 heating precautions 97 injuries from 101 making in the laboratory 33. 248. 215 urogenital specimens 207–208. 297–298 erythrocytes 279–288. 224–228. 36–37 autoclaving 87. 259. 280–283. 236 Glucose reagents 358 Glycerol–malachite green solution 359 Gonococcal infection 197. 313 Habitat. 39–42 stills 25. 320 microscopes 32 registers 47. 172 malaria parasites 173 Giardia intestinalis 115. see Human immunodeficiency virus Hookworm (Ancylostoma duodenale/ Necator americanus) 106 adults 148. 258–259 Fungi 204. 39–42. urine 243. 317 specimen collection 267–270. 133. see Gonococcal infection Graduated conical glasses 77 Graduated pipettes 73. see Fluorescent treponemal antibody-absorbed test Fuchs–Rosenthal counting chamber 258. 158 Hot-air ovens 33. see Spectrophotometers Haemophilia 298. 271–279. 323–324 CSF specimens 261–262 substance concentration 7 urine specimens 236. 261 Hair fungal infections 225–226. 121. see Urogenital specimens Geographical distribution filarial parasites 160. 49 reticulocytes 316–318. 126–144. common filarial parasites 165 Haematology (see also Haemoglobin) 265–321 blood cell types 265–266. 154 Formol gel test. 265–266 Haematology (continued) blood clotting 297–298. tests 342–344. 89–90 Household bleaches 84–85 Howell–Jolly bodies. 253 Granular casts. 128. 275. 199–201 acetone–ethanol decolorizer 351 CSF specimens 260–261. 342 . 263 Glucose blood concentrations 322–325. 26 storage 45 tubing 37. 39–42 Globulins. precautions 97 Generators. 286–287 ESR 292–295. 304. 156–159 adults 146–152. 252. immature 313. 208. electrical 18. 309 b-Human chorionic gonadotropin (b-hCG) 339 Human immunodeficiency virus (HIV) 341–342. 73 Gram staining 199–201. 146. 344 Heterophyes heterophyes 106. 139 HIV. 158 transmission routes 106 Heparinized tubes 42 Hepatitis. 39–42. electrical supply 13 Genital specimens. 152 larvae 157. 70 Hansen disease. 298 Haemophilus influenzae 257. 261. 125. 272–273. 146 urine 240. 306–314 thrombocytes 321 Haemoglobin erythrocyte relationship 284–285 estimation 7. 147–151 eggs 125. 71 Heinz bodies 319 Helminths intestinal 105. 253 Fuses. 260–261 urine specimens 252–253. reagents 350 FTA-Abs. 261. 226. 125–152. 261 identification 124–125. 129–144 larvae 156–159. 121 Giemsa stain 175–177. 278. 87 cleaning 77–81. 293–294 leukocytes 319–321. 253 Gonorrhoea. 227 specimen dispatch 93 Hand-operated centrifuges 70. see Leprosy Harada–Mori sedimentation technique 156–157 b-hCG. CSF (Pandy test) 262–263. 267–270 test equipment 37. 150 eggs 126. 127. 305. 243–244 Granulocytes. 300–304. centrifuge 70. 42–43 thin blood films 299–314. see b-Human chorionic gonadotropin Heads. 128. 18 Gas burners. erythrocytes 309. 357–358 Glassware 34–35. 129–151. 115. 248. leishmaniasis 196 Formulae. 165.

169. 129–144 larvae 156–158. 159–161 transmission routes 106 Inventories. 146 helminths adults 146–152. 120. urine 243. 122 cysts 118–124. 126–144. 179–181 Trypanosoma spp. 120 ciliates 116. 144–145 Yersinia pestis 204 Immunofluorescence 332–333. 239–240 Labelling biopsy specimens 96 . 171–172 fungi 124–125. 151 Hydrochloric acid 359 Hygiene. 121 Blastocystis hominis (yeast) 124–125. 147–151 eggs 125. 193–194 Corynebacterium diphtheriae 201. 114. 313 Hypochlorite solutions 84–85 Identification Bacillus anthracis 204. 158 tapeworms 149 intestinal protozoa 111–117. laboratory waste 90. see Antibodies Immunology 328–349. 195–196. 329 antigens 329–330 immune system 328. 121. cleaning and maintenance 83 Infants (see also Children. 121 sedimentation techniques 153–157. 139. urine specimens 239–240. 135–137 reticulocyte number concentration 318 stool specimens 217 urine collection 234 Infection sources African trypanosomiasis 183 blood/skin parasites 160 Chagas disease 193 intestinal parasites 106 leishmaniasis 194 Infectious mononucleosis 313 Injuries. 120 ciliates 116. 152 Hypersegmented polymorphonuclear neutrophils 313. 119–122. 123 Iron deficiency 288 Isospora belli 124 Isotonic saline. 196 leukocytes 125. 173–182. intestinal parasites 106 Hymenolepis spp. 174–176. 121 coccidia 122. 129–144 larvae 156–158. 124–125 amoebae 113–114. 187. 107–111. 124–125 flagellates 115–116. 121. 198 Instruments 32 disinfection 84 storage 45 Intestinal parasites 105–159 concentration techniques 152–156. 149 eggs 128. 121. 146 pus 125. 171–172 Plasmodium spp. 121. 115–116. 162–163. 147–151 eggs 126–144. 152–157 helminths 125–152.Index 375 Hyaline casts. 90–91 Incubators. 120. Neonates) capillary blood collection 280 erythrocyte number concentration 287 Infants (continued) erythrocyte volume fraction 284 leukocyte number concentration 291 leukocyte type number fraction 320 normal haemoglobin 275 normal thrombocyte counts 321 pinworm egg collection 135–137. 166. 142–143 Ketone bodies. 158 protozoa 111–117. 129–151 adults 146–152. 329 immunochemical techniques 330–335 tests 336–349 Incineration. 121 Iodine 85. 243 Hydatid cysts (Echinococcus granulosus) 151. 117. 124 amoebae 113–114. 119–122. 329–336 antibodies 328–329. 154–156 stool specimens 107–110. 185–194. 121 Leishmania spp. supplies 46 Iodamoeba butschlii 114. 166–167. 111–124. 125 starch granules 144. 332 Immunoglobulins. 204 Balantidium coli 116. laboratory accidents 98–101. 115–116. 118–120. 125 blood parasites microfilariae 163–172. 167. 113–115. 99 Inoculating loops 197–198. 119–122. 139. 125. 202 filariae 159–172. see Sodium chloride Kahn tubes 34 Kato–Katz technique. 125 pollen grains 146. Schistosoma mansoni infection 141–143. 122 flagellates 115–116. 118. 121 coccidia 122. (tapeworm) adults 148. 121. 111–125. 113–115.

360 Lugol iodine 360 Lutzomyia longipalpis 194 Lymphatic system. 291. 73–77 Litre. definition 3. 139 Meters. 255–257 malaria 180–182 number concentration 6. 288–292 type number fraction 6. 293–294 helminth eggs 126–127 liquids 73–77. 242–243 Leukocytosis 291 Leukopenia 291 Liquids heating precautions 97 measurement/dispensing 73–77. 354. 314 Lymphocytes 311–313. 16 Methylene blue solution 361 Metric system 2–7 Microcytes 307. 63–64 pH. aspiration 183–185. 313 Leukocytes 125. 165. 159–160. 307 Microfilariae 159–172. 360 Mean erythrocyte haemoglobin concentration 285–286 Measles 312. 265. 184–185 Lymphoblasts 313. 59 Lancet flukes (Dicrocoelium spp. 172–182. 242.376 Index Labelling (continued) specific reagents 350–368 specimens for transport 94 Laboratory workers. 209–210 Mammomonogamus laryngeus 205 Mancini technique. 311–313 Lymphocytosis 320 Lymphopenia 320 Lysis time. 171–172. 172 Loeffler methylene blue 204. 345–346 Male patients. 265. 295–296 blood clotting 297–298. 178. 221–223 Leukaemia 285. 259 common causes 256 CSF examination 197. 320 dipstick test 344–346. 308 Macroscopic examination. 67–69 bleeding time 295–296. see Direct examination Madura foot. microscopes 53–56. 259. 134 Lancets 36 Larvae (see also Microfilariae) 156–157. 235–236 protozoa cysts 123 quantities 2–3 traditional system 5–7 Megakaryocytes 266. responsibilities 2 Lactophenol cotton blue mounting solution 359 Lamps. 163. 265. 288– 290.) 159. 5 May–Grünwald stain 299. 159–160. 309 Leishman stain 299. electricity 16. 314 Meker burners 35 Meningitis 255. 73–77 lysis time 297–298. 297–298 metric system 2–7 ocular micrometers 63. 304. 319–320. 54 Leprosy (Mycobacterium leprae) 220–224. 313. 66–69. 65 water-baths 83 Malaria (see also Plasmodium spp. 336. 261. 311–314 CSF specimens 255–257. 139. 314. 309 blood cells 312. 207–208. filarial worms 159 Lymph nodes. 163. microscopes 53–54. 359 Leishmaniasis (Leishmania spp.) 106. 305. 297–298 ESR 292–295. 310–314. 291. 134. 260–261. Number concentrations. Number fractions) 2–7 balances 32. 303. blood specimens 297–298. 263 Meningonema spp. 159 Mercurothiolate. 307 Liver flukes. 163. 196 Lenses. see Flukes Loa spp. 297–298 Lysol 84–85 Macrocytes 307. urine 235. 125.) 194–196. 338 Lead batteries 14–15 Lead poisoning 255. 128. 249 . microscopes 58–60. 305. 259. see Radial immunodiffusion Mansonella spp. 320 Measurement (see also Calculations. 166. 263 tuberculous 257. see Mycetoma Magnification. 320 urine specimens 240. 260–261. 165. 163. measurement 4–5 Liver diseases 306. 313. 354. 54–56 Maintenance (see also Repairs) batteries 15 burettes 77 centrifuges 83 incubators 83 lead batteries 15 microscopes 64–66. 158 stool specimens 109 urine specimens 248 Latex agglutination techniques 336. see Thiomersal Metagonimus yokogawai 106. hepatitis B virus 342–343 Mass. urogenital specimens 197. 172 Markers. 128.

212–215 sickle-cell anaemia 315. 209–210 Gram-stained organisms 200–201. 165. 229. 59 maintenance 64–66. 178–182 microfilariae in blood 164. 229 protozoa cysts 124. 312 Nail specimens. 264 Neonates (see also Children. 79–80. 152 larvae 157. 251–254 vaginal discharge specimens 215 watery stools 216. 217 Yersinia pestis 204 Mole definition 3 substance concentration 5 Molecular weight 3 Monocytes 311–312. 259. 288–291. 80 Microscopic examination acid-fast bacilli 203 Bacillus anthracis 204. 57–58 cleaning 64–65. 145 Onchocerca volvulus 160–163. ocular 63. stools 157–159. 310 Number concentrations 5–7 erythrocytes 265. 319–321 reticulocyte 6. 140. 171–172 microfilariae. 315–316 sputum/throat specimens 206 stool specimens 107–109. 223 Necator americanus (hookworm) adults 148. 226. 150 eggs 126. 62–63 illumination system 56–57. 162–163 mycetoma pus specimens 227 Mycobacterium leprae 223.Index 377 Microhaematocrit equipment 37 Micrometers. Infants) erythrocyte number concentration 287 erythrocyte volume fraction 284 immune system 328 leukocyte number concentration 291 leukocyte type number fraction 320 normal haemoglobin 275 Nephelometry. 200–201. 221–223 tuberculosis 197. polymorphonuclear 310–311. leprosy 220. 227. 59–63. 226. 124 . spermatozoa 214 Mott cells 259. 317 Occult blood. 211 thin blood films 305–314. 288–290. 311. 195–196. 166–172. 87 Neisseria meningitidis 260. 258–261 fungi 226. 310. 158 Needles disposable 36 reusable 81. pus examination 226–227 Mycobacterium leprae 202. 316–319. 241–248. 223–224 pityriasis versicolor 229. 321 Number fractions 5–6 leukocyte type 6. 222. 140. 287–288 leukocytes 265. 63–64 Oil droplets. 249 Oocysts. 288. 63–64 Micropipettes 38–39. 159 Ocular micrometers 63. 65 mirrors 56. 319. Cryptosporidium spp. 65 adjustment system 57. 38. dispatch 93 Nasal specimens. 74. 206 Mycology (see also Fungi) 225–229. Mycobacterium leprae 224 Mortars/pestles 35 Motile forms (see also Trophozoites) protozoa 111–117. 223–224. 312 Morphological index. 285. 204. 185–187. see Nucleated erythrocytes Nucleated erythrocytes 292. 317–318 semen specimens 212–214. 123–124. 161–163. 53–66. 59. Onchocerca volvulus 162–163. 258–259 reticulocytes 316–319 thrombocytes 266. 220–224. 290 Neutral red 361 Neutropenia 320 Neutrophilia 320 Neutrophils. 260. 56. identification 145. 53–57. 313. 166–167. 260 Mycetoma. cleaning 77 Nickel–cadmium (Ni–Cd) batteries 15 Normoblasts. 128. 81. 74–75 Microscopes 32. 313 New glassware. 288–292 leukocytes in CSF 258–259. 56–57. 253 Leishmania spp. 60. 60 precautions 65–66 setting up 58–61. 229 gonorrhoea 208. 185–188 urine 241–248. immunology 335 Nephrotic syndrome 247 Neubauer counting chambers 259. 220 body cavity fluid 219–220 Corynebacterium diphtheriae 202 CSF 257–261. 65 focusing 61. 87. 196 leukocyte type number fraction 319–320 malaria parasites 178. 169. 250–253. 59–61 slides 37. 306–314 thrombocyte number concentration 321 Trypanosoma spp. 284. 228–229 Myelofibrosis 309 Myeloma 237. 108–109 syphilis treponemes 211. 124–125 reticulocyte numbers 317. 111–118 Motility.

186–187. 135–137 Pipettes 37–39. 39–40. 93–94 Pandy reagent 361 Pandy test. 328 erythrocyte volume fraction 279– 280 glucose concentration 325 serum comparison 266 Plasmodium spp. 67–68 Opisthorchis felineus. 248. 209 Pathogenicity. electrical 18–19. 196 sputum/throat 205 transmission routes 106 urine 240. 140 Ordering. SI 4 Preparation (see also Slide preparation) reagents 350–368 smears 197–199. immunology 334–335. intestinal protozoa 111 Pelvic cells. 49–51 leishmaniasis 194–196 skin 159–163. 89–90 Packed cell volume. 110 urine specimens 234 Pressure cookers. hot-air 33. see Thrombocytes Plugs. 159. 313 Polyvinyl alcohol (PVA) 110. 335 Precipitin tubes 34 Prefixes. 18–19 Plumbing 20–23. electrical 17 Precautions (see also Safety) accident prevention 97 anthrax specimens 204. 171–172 protozoa 172–196. 135–138. 263 Panels. 128. 196 malaria 172–182. CSF globulin 262–263. 140 Parasites (see also Intestinal parasites) 105–196. 187. 262–263 urine specimens 236–239 . specimens for dispatch 91–94. 140. 184–187. 193–194 trypanosomiasis 182–194. 300–304. 146 Plasma abnormal proteins 298 Plasma (continued) cells 312. supplies 46 Orthostatic proteinuria 237 Ovens. 116 thin blood films 303 Precipitation. 173–182. 185. eggs 128. 39–40. (see also Malaria) 124. 248–251. solar 13–14 Paragonimus westermani 106. 163. 152–156 laboratory registers 47. 242 Pestles/mortars 35 Petri dishes 34. 73–75 cleaning 78–79. 320 blood 159–160.378 Index Open two-pan balances 67–68. 178. 313. 161–163. 178 microfilariae 159–172. urine 235. laboratories 11–12. urine 242. 251 Pasteur pipettes how to make 33. 146 Polycythaemia 287 Polymorphonuclear cells 310–311. 128. 198–199 thin blood films 299–314. identification 146. 11–12 Plant parts. 345–346 Platelets. 163–194. 306–314 Preservation stool specimens 109–110. 309 Poisoning laboratory accidents 100 lead 255. equipment sterilization 88–89. 39–40 precautions 97 Pityriasis versicolor (Pityrosporum furfur) 227–229. 175–176. 356 potassium hydroxide 225 stool specimens 107. haemoglobin estimation 271–279. 189–192 concentration techniques 152–156. 311. 272–273 Pinworm (Enterobius vermicularis) adults 146. 37 Phenol red 361 pH measurement. identification 145–146. 312. see Erythrocyte volume fraction Packing. 235–236 Phosphate-buffered water 361–362 Photometers 32 Photometric methods. 309 Pollen grains. 195–196. 20–23 Poikilocytes 309. 178 dipstick test 344–346. 362–363 Potassium cyanide 274 Potassium hydroxide 363 Potassium permanganate 363 Power. 220 anticoagulants 43 autoclaving 88 centrifuges 72 CSF specimens 255–256 electricity 19–20 ELISA for hepatitis B 343–344 hypochlorite solutions 85 microscope care 65–66 pipette use 75 potassium cyanide 274. 140. 87 urogenital specimens 209. 178. 189–194. 166. 79 Pasteur 33. 228–229 Plans. 89 Prevention accidents 97 laboratory infections 96–97 Propane gas burners 97 Protein CSF specimens 262–263. 73–75. 39–40 sterilization 87. 147 eggs 126.

52 Pus casts. modified 365 sulfosalicylic acid 365 thiomersal–iodine–formaldehyde (TIF) 365–366 o-toluidine 322–325. 368 Wintrobe solution 368 Zenker fixative 368 Ziehl–Neelsen stain 202–203. 347–348. 93–94 Reference ranges blood glucose concentrations 324–325 . syphilis 346. laboratory 38. 47–52. 111–117. 351 alkaline haematin D reagent 276. 354 Records. 366–367 Wayson stain 203. 125 mycetoma 227–228 PVA. 350–368 acetic acid 350–351 acetone–ethanol decolorizer 351 acid–ethanol for Ziehl–Neelsen stain 351 acid reagent 351 Albert stain 201. 335. 367 Willis solution 152. 178.Index 379 Protozoa blood 173–194. 193–194 intestinal 105–106. 187. see Quaternary ammonium compounds Radial immunodiffusion 335. 223–224 Red blood cells. 119–122. urine 243. 244 Pus specimens Bacillus anthracis 219–220 containers 83 CSF 256. pH 7. 185. 360 Lugol iodine 360 May–Grünwald stain 360 methylene blue solution 361 modified Schaudinn fixative 362 neutral red 361 Pandy reagent 361 phenol red 361 phosphate-buffered water 361–362 polyvinyl alcohol (PVA) fixative 362–363 potassium hydroxide 363 potassium permanganate 363 safranine solution 363 saponin 363–364 silver nitrate 364 sodium bicarbonate 364 sodium carbonate 364 sodium chloride 364 sodium hydroxide 364–365 sodium metabisulfite 365 streptolysin O 336–338 Stuart transport medium. 124 transmission routes 106 Public health. 111–124. 351. laboratory reports 47–48. trophozoites 117 Rapid plasma reagin (RPR) test. 368 fluoride oxalate anticoagulant 357 formaldehyde 357 Giemsa stain 357–358 glucose reagents 358 glycerol–malachite green solution 359 hydrochloric acid 359 lactophenol cotton blue mounting solution 359 Leishman stain 359 Loeffler methylene blue 204. 348 Reagents 1.2 29–31. 219. 365–366. modified Hucker 355 Delafield’s haematoxylin stain 355 diacetyl monoxime stock solution 367 dichromate cleaning solution 355 Reagents (continued) disodium hydrogen phosphate stock solution 361–362 Drabkin diluting fluid 355–356 EDTA dipotassium salt 356 eosin 356 expiry dates 46 Field stains 117. 354 Carbol fuchsin solution 354 Cary–Blair transport medium 354 chemical formulae 350 colour reagent 367 crystal violet. 330 Rapid Field stain. 358 trisodium citrate 366 Türk solution 366 urea reagents 325. 323–324. specimen dispatch 91–96. measurement 2–7 Quaternary ammonium compounds (QUATS) 85 QUATS. 339–341 Radioimmunoassay 330. 356–357 fixatives 95. see Polyvinyl alcohol Pyelonephritis 237 Quality assurance 101–102 Quality control AHD reagent 352 demineralized water 28–29 distilled water 27 Quantities. 362–363. 221. 210 identification 125. 256 dispatch 92 gonorrhoea 210. see Erythrocytes Reference laboratories. 352 Amies transport medium 352–353 Benedict solution 353 benzoic acid 358 blank reagent 353 boric acid 353 brilliant cresyl blue 353 buffered glycerol saline 353–354 buffered water.

see African trypanosomiasis Slide preparation (see also Thick blood films. 250–251 Sedimentation rate. erythrocytes (ESR) 292–295. 20–23 Reports. 306–310 fungal infections 225–226. see Rapid plasma reagin Safety (see also Precautions) electricity 19–20 in the laboratory 97 pressure cookers 89 Safety bulbs. 310. 248. 212–214 Serological tests African trypanosomiasis 188–192. 251. 17–20 microscopes 65 plumbing 20–23. 47–48. 80 supplies 37 Slit skin specimens cutaneous leishmaniasis 195–196. 249–251. 221–223 parasites 159–163. 242–243 Renal disease 236 Repairs (see also Maintenance) electrical equipment 17–19. 315 Silver nitrate 364 Sink traps. erythrocyte volume fraction 280–281 Scalpels 36 Schaudinn fixative. 251. 253 Reticulocytes 6. 127–128. 260–261. 198–204. plumbing 22–23. 287 ESR 293–294 leukocytes 291. 316–319. 140–141. 307–308. haemoglobin 272–273. Thin blood films) 198–201. 250 Semen specimens 211–215. 87. 307. 223 malaria diagnosis 173–179. 253 Resolving power. 81. 248. 161–163. 260–261 faecal 141–143. 310 test 314–316. determination 336. 87 Rheumatoid factors. laboratory 38. 196 Sleeping sickness. 87. 24 Sanitation. 196 leprosy 221–222. 272–273 Refrigerators 32 Registers (see also Records) specimens 47–51 Renal cells. 228–229. 229 gonorrhoea diagnosis 208 leprosy diagnosis 222–224. reporting 47–48. modified 362 Schistocytes 308. 250–251 mansoni 141–143. 87 Reusable syringes 81. 175–176 semen specimens 212 sputum/throat specimens 206 syphilis diagnosis 210 urine specimens 252. 133. 320 normal haemoglobin 275 reticulocytes 318 thrombocytes 321 Reference solutions. (blood flukes) 108. 52. 150–151. 20–23 Saponin 363–364 Scale readers. urine 242–243. 222 Onchocerca 161–162. 133. plumbing 20–23. 189–192 registers 47. 51 Serum glucose concentration 325 plasma comparison 266 Sharps containers 96 Shocks. 223 . 240. 195–196. 248.380 Index Reference ranges (continued) blood urea concentrations 327 erythrocytes 284. 142–143 spp. 147 eggs 126. 317 Reusable needles 81. 22–23 Sizes. 142–143 Mycobacterium leprae 220–225. electric 101 SI units (International System of Units) 2–7. 308 Schistosoma eggs 128. 336 Ringworm 225–226. 152 RPR. rubber 35 Safranine solution 363 Sand filters 24. helminth eggs 132 Skin acid/alkali splashes on 98. laboratory workers 2 Results. 310. 271 Sickle-cell anaemia 292. 293–294 Sedimentation techniques 153–157. 52. 204 anthrax diagnosis 220 body cavity fluids 218–219 erythrocytes 305–310. 252 vaginal discharge specimens 215 Slides cleaning 79–80. 161–162 Smears CSF 259. 226. 226. 151 Schistosomiasis (see also Schistosoma) 320 urine detection 234. 299 Roundworm (Ascaris lumbricoides) 106 adults 146. 249–251. 228–229 Mycobacterium leprae 220–224. 100 disinfection 84 Skin specimens (see also Slit skin specimens) dispatch 93 fungal infections 225–229. 226 Romanowsky stains 265. 140–143 haematobium 248. 154–156. microscopes 55 Responsibilities.

124 faecal trophozoites 117–118 Leishmania spp. 238. 164. 175. 353 Delafield’s haematoxylin 355 eosin 117–118. 305. 42–44. volumetric flasks 76 Storage supplies 45 water 24 Streptococcus pneumoniae 257. 356 Field 117. 26–27 Specimen collection appropriate 102 blood 267–270. 86–88 boiling 89 dry heat 89–90. 304. Disinfection) 85–90. 184–185 pinworm eggs 135–137. 195 microfilariae 170–171. 171–172 Onchocerca volvulus 163. 40 Stocktaking. 267–270. 354. 228 leprosy 221–222. disinfection 85 Stool specimens containers 42. 317. 286 malaria parasites 174–175. 299 Wayson 367 Standard curve. 318–319. 199–204. electrical supply 13–15 Solar stills 26. haemoglobin concentration 273 Starch granules. 196 sputum 205. 189. 304 faecal cysts 123–124. 163 Plasmodium spp. 177 smears 199–201. 252 urogenital samples 207–208. 227–228. 262. 135–137 pus 220. 273. 201–202 dispatch 92 Staining techniques 109 blood films 175–176. 107–110 watery 216–218. 304. 260–261 Streptolysin O 336–338 Strongyloides stercoralis eggs 109. 205–206 stools 107. 301 trypanosomes 186. 42. identification 144. 217 Stoppers. 174 microfilariae 164. 143 . 255 fungal infection 225. 175–178. see Trisodium citrate Sodium dihydrogen phosphate. 263 Spirit lamps 35 Sputum. 303–305. 152–153 Sodium citrate. 93–94 disposal 97 registration 46–51 Spectrophotometers (see also Colorimeters) 271–279 calibration 272–274. disinfection 84 Sputum specimens 204–206. 189 body cavity fluids 218 CSF 255. 227 semen 212 skin 161–163. 280–282. 305. 249. 356–357 Giemsa 299–300. 91 using a pressure cooker 89–90. 195. 360 Romanowsky 265. 199–204. 221–223 lymph node aspirates 184–185. 351 brilliant cresyl blue 316–317. 159 parasitology 107–110. 145 Sodium bicarbonate 364 Sodium carbonate 364 Sodium chloride 364 flotation technique 152–153. supplies 45–46 Stools. 260. 186. 89–90 autoclaving 86–87. 281. 201–202. 82 Specimens dispatch 91–96. 143. 357–358 Gram 199–201 Leishman 359 May–Grünwald 299. identification 145. 205 containers 44. 25–26 Stirring rods. hereditary 308 Spinal compression 257. 90 Stills demineralized water 33 distilled water 24–27. 217 syphilis chancre 210. 198–199 staining techniques 199–201. 308 Spherocytosis. 82. 86–87. Ziehl–Neelsen stain) Albert 201. 166 thin blood films 300.Index 381 Smears (continued) preparation/fixation 197–199. 42–44 cleaning 81–82. see Sodium bicarbonate Sodium hydroxide 364–365 Sodium hypochlorite 84–85 Sodium metabisulfite 365 Solar energy. 204 Soaps. 82 Sputum specimens (continued) Corynebacterium diphtheriae 201. 210 throat swabs 206. 215 Specimen containers 34. 144–145 Sterilization (see also Cleaning. 206 urine 231–232. 204 Staining troughs 34 Stains (see also Staining techniques. 216–217. 128. 241. 82 dispatch 92 occult blood 157–159. 161–163. 276–277 Sperm counts (see also Semen specimens) 214–215 Spherocytes 308. anhydrous 361–362 Sodium hydrogen carbonate. 44. how to make 40–41.

304 . 154–156 semen examination 212. 208 leishmaniasis diagnosis 196. 307 Techniques (see also Tests) agglutination 188–192. 108–109 syphilis diagnosis 210–211. 147. 152 identification 149 Tally counters 37 Tapeworm. see Units. 264. 338 leishmaniasis formol gel 196 Onchocerca volvulus 160–163. 263 Sulfosalicylic acid 365 Sulfuric acid. 81. detection 173–182. 143–144. 220 antibody determination 339–341 ASOT 336–338 Chagas disease 193–194. 175–177 cellophane faecal thick smear 141–143. 209. 226. 261. 175 Trypanosoma spp. 173–182.382 Index Strongyloides stercoralis (continued) larvae 156–157. 315 stools occult blood 157–159. 204. 142–143 sedimentation 153–157. 20–22 Target cells. 149 eggs 128. 262. 166–167. 189–192 anthrax 204. 336. water supply 20–22. 134. 184–187. 333–334. 37 cleaning 82 heating precautions 97 holders 35 Thalassaemias 306–309 Thermometers 35 Thick blood films Chagas disease 193–194. 300–303 staining 303–305. 171–172 oocyst detection 123–124. fish (Diphyllobothrium latum) 106. 193–194 Plasmodium spp. 87 Système internationale. 306–314 Plasmodium spp. 338–339 anthrax detection 204. 143–144. 36–39 ordering procedures 46 stocktaking 45–46 Supplies (continued) storage 45 water 24–31 Switches. 178 Schistosoma mansoni detection 141–143. 256–264 fungi detection 225–229. 169. 336 sickle-cell anaemia 314–316. (tapeworm) 106 adults 146–148. 261 gonorrhoea diagnosis 207–209. 343–344 falciparum malaria 344–346. 153–154 Tests (see also Techniques) African trypanosomiasis 188–192. 128. 345–346 hepatitis 342–344. 147–148 thin blood films 300–304. 189–192. 330–335 latex agglutination 336. 365 Subarachnoid haemorrhage 256–257. 257. 144. 211 tests 346–349. 342 immunology 330–335. 124 parasite concentration 152–156. 147–149 eggs 128. see Thiomersal Thin blood films 299–314. 161–163 rheumatoid factors determination 336. 189–194 urine protein estimation 238–239 Willis solution flotation 152–153. 164–172. handling 97 Supplies electricity 12–20 laboratory 33. 344 HIV 341–342. 81.) adults 146–148. 152–157 Techniques (continued) pinworm eggs examination 135–138. detection 183–194. 173–182. 348–349 Syringes 36. SI Taenia spp. 264. 204 sputum/throat swab examination 205–206. 152 Tapeworm (Taenia spp. 34–35. 221–224 microfilariae detection 164. 300–304. 199–204. 306–314 trophozoites staining 117–118 Trypanosoma spp. 187 Thimerosal. 142–143 CSF examinations 256–264. 134. 135–137 Plasmodium spp. 209–211. 228–229. 158 Stuart transport medium 207. 87. 204. 19 Symptoms African trypanosomiasis 183 Chagas disease 192–193 leishmaniasis 194 malaria 172 Syphilis (Treponema pallidum) 197. 348–349 Test-tubes 34. 219–220 biopsy fixation 95–96 blood sample staining 175–176. 193–194 ELISA 341–342. 333–336. 205–206 stools examination 107–109. 187. 336. 195–196 leprosy diagnosis 221–223. electrical 19. 175 preparation 300–303. 346–349 tapeworm detection 147–149. erythrocytes 306. 300–314. 211. 158 syphilis infection 346–349. 152 identification 149 Taps. 144. 212–214 smear staining 199–201.

252–253 blood 234. 239–240 pH measurement 235. urine 245. 182–194. 259 CSF examination 263 Ziehl–Neelsen staining 261 Tubes (see also Test-tubes) centrifuge 72. 295 Tuberculous meningitis 257. 264. 320 Units. 358 Tongue depressors 36 Tools electrical repairs 17. 116 vaginalis 215. 250–253 abnormalities detection 241–248. 113–115 ciliates 116. 365 Treponema pallidum 209–210. 245 Urine disinfection 84 neutral 245–246. 246. 128. 241–248 analysis register 47. 110.) 161. 259. blood urea concentrations 325–327 Threadworm. 247 Urea 7. 152 Triple phosphate crystals. see Dispatch Transport media 263–264 Amies 352–353 buffered glycerol saline 218 Cary–Blair 216–217. see Thiomersal–iodine– formaldehyde Timers 35 Tinea infection 225–226. SI 2–7 Urates. 39–40. 346–349 pertenue 210–211 Trichloroacetic acid 322–323. 236 b-hCG determination 339 ketone bodies 239–240. 266 deficiency 298 number concentration 7. 245 glucose detection 236. 358. 241–242 casts 243–244. 209. 353. 106. 128. 321 TIF. 248 Urine specimens 231–254. 39–42 Westergren 37 Turbidimetry. 17 laboratory 38 plumbing 20. 93–94 lead batteries 14 specimens. 249 Trichostrongylus spp. 226 o-Toluidine reagent 322–325. 241–243 containers 44. urine 246. 37. 319. 185. 235–236. 235–236 preservation 234 protein detection 236–239 Schistosoma haematobium 248. 268 Toxoplasma gondii 124 Traditional system. 82 crystals 236. 250–251 Urogenital specimens gonorrhoea 207–208. 184–187 Chagas disease 192–194. 241–242. urine 245. leukocyte 6. 248. 144. see Pinworm Throat specimens 92. 49 bacteriological examination 251–254. 325–327 Urea reagents 325. 34.Index 383 Thiomersal–iodine–formaldehyde (TIF) 109. 323–324. 354 Stuart 207. 117 flagellates 115–116. 20 Tourniquets. 144 Trichuris trichiura (whipworm) 106 adults 150. 115–116 staining methods 117–118 urine specimens 240. 253 Tuberculosis 197. blood collection 268. immunology 335 Türk solution 366 Type number fraction. 111–118 amoebae 113–114. 208–209 Thrombocytes 266. 245–248. 150 eggs 126. 210 vaginal discharge 215–216 Vacuum pumps 79. 366–367 Uric acid crystals. measurement 5–7 Transformers. 247. 72 glass 33. 260 Trypanosomiasis (see also Trypanosoma spp. 249–251. 144. 193 Tubercle bacilli 253. 206–209. 245–248 dispatch 93 foreign substances 244–245. 193 CSF 259–260. 246 Trisodium citrate 366 Trophozoites 111–117. 208 syphilis 209–210. 264. 249 Trypanosoma spp. 366 Trichomonas hominis 115. 295 African 183–192. 187. 79 Vaginal discharge specimens 215–216 Vectors (see also Transmission) common filarial parasites 165 . 144. 159. 365–366 Thiosemicarbazide. electrical 17 Transmission African trypanosomiasis 183 blood/skin parasites 160 Chagas disease 193 intestinal parasites 106 leishmaniasis 194 Transport biological specimens 91–96. 239–248. 243–244 cells 241–243. 319–320 Typhoid fever 291.

76 Volumetric pipettes 73–74. definition 3 Westergren tubes 37. 74 Wall sockets. 165. 83 Whipworm (Trichuris trichiura) 106 adults 150. 218. Helminths Wuchereria bancrofti 248. 280–283. 313. laboratory balances 66–69. 249 spp. 211 Yeasts 124–125. 344 Visceral leishmaniasis 196 Voltage. 42. 281. 368 Wintrobe solution 368 Work benches. 217 Vincent’s bacilli 199. see Leukocytes Willis solution 152–153. 150 eggs 126. 159–160. 75–76. 144. 37. 321 diarrhoeal disease 105 hepatitis 342–344. 354. 201. erythrocyte 6. 144. 37 Water laboratory supplies 23–31. 83 Watery stool specimens 216–218. 219 Zenker fixative 368 Ziehl–Neelsen stain 123–124. 201 Virus infections 312. 90–91 Watch glasses 34. 267–270. 202–203 acid–ethanol 351 carbol fuchsin solution 354 smears 221. CSF 257. 41–42 Waste disposal 90–91.384 Index Venous blood collection 267–270. disinfection 85 Worms. 217 Wayson stain 203–204. 125. 286 Volume. electrical 19 Wash bottles 35. 67–69 Weight. 286 examination 166–170 glucose concentration 325 Vibrio cholerae 216. 361–362 plumbing 20–23 waste 22–23 Water-baths 32. 152–153. 128. 163. SI derived units of 4–5 Volumetric flasks 34. 152 White blood cells. 222–223 tuberculous meningitis 261 . 204 Yersinia pestis (bubonic plague) 218 staining techniques 203–204. 257 Yaws (Treponema pertenue) 210–211. 367 Weighing. see Filariae. 172 Xanthochromia. 279–287. electrical supply 16 Volume fraction.

1993 (133 pages) Basics of quality assurance for intermediate and peripheral laboratories. Van Dyck E. 1999 (244 pages) Safety in health-care laboratories. 2 2002 (256 pages) Further information on these and other WHO publications can be obtained from Marketing and Dissemination. Giroult E. No. WHO Regional Publications. and hospital equipment.1) 1997 (157 pages) Laboratory biosafety manual. 1991 (122 pages) Basic laboratory methods in clinical bacteriology. World Health Organization. 1211 Geneva 27. Piot P. diagnostic imaging. (document WHO/LAB/97. Rushbrook P. . El-Nageh MM et al. Meheus AZ. Switzerland. 1999 (146 pages) Maintenance and repair of laboratory. 1991 (128 pages) Laboratory diagnosis of sexually transmitted diseases. eds. 2nd ed. Prüss A. 1994 (164 pages) Safe management of wastes from health-care activities. Eastern Mediterranean Series. 2nd ed.Selected WHO publications of related interest Basic laboratory methods in medical parasitology.

measurement and dispensing of liquids. ISBN 92-4-154530-5 9 789241 545303 . are scarce and the climate is hot and humid. including equipment. Methods of disposal of laboratory waste. The first describes the setting-up of a peripheral health laboratory and general laboratory procedures. bacteria and fungi. the book emphasizes simple. dispatch of specimens to reference laboratories and laboratory safety are also discussed. Techniques for the preparation. The third and final part describes the examination of urine. including techniques based on immunological and serological principles. followed by a detailed description of the method and the results of microscopic examination.This manual provides a practical guide to the safe and accurate performance of basic laboratory techniques. A summary of the reagents required for the various techniques and their preparation is provided in the annex. Intended for use by laboratory technicians working in peripheral-level laboratories in developing countries. protozoa. The book is divided into three parts. a list of materials and reagents is given. disinfection and sterilization of laboratory equipment. including use of a microscope and laboratory balances. centrifugation. The second part describes techniques for the examination of different specimens for helminths. cerebrospinal fluid and blood. fixation and staining of smears are also discussed. Numerous illustrations are used throughout the book to clarify the different steps involved. For each technique. economical procedures that can yield accurate results where resources. and cleaning.

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